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Enzymes: Biochemistry

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A reaction may be thermodynamically favored, but ____________ ___________.
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Extremely slow Not a useful time scale for the reaction to occur.
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Enzymes
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1. Highly specialized molecules that have high catalytic power. 2 High degree of specificity for substrates 3. They accelerate chemical reactions
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T/F: All enzymes are proteins.
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False; All enzymes are proteins, WITH THE EXCEPTION of a small group of catalytic RNA molecules. Not ALL enzymes are proteins
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What is essential for a protein’s enzyme catalytic activity?
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A proteins primary, secondary, tertiary and quaternary structures of protein enzymes are essential for their catalytic activity. 1. If an enzyme is broken down into its components, denature or dissociated into subunits, the enzyme catalytic activity is destroyed.
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Simple enzyme
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A simple enzyme is an enzyme that requires no chemical groups for their catalytic activity, only their AA residues.
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Cofactor
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An enzyme that requires an additional chemical component for catalytic activity of 1) one or more inorganic ions (Fe2+, Mg2+, Mn2+, Zn2+)
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Coenzyme
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An enzyme that requires an additional chemical component of catalytic activity of: 1) A complex organic or metalloorganic molecule.
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Some enzymes require BOTH a coenzyme and one or more metal ions for activity (cofactor).
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Prosthetic Group
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A coenzyme or metal ion that is TIGHTLY or even covalently bound to the enzyme protein.
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What is another name for a prosthetic group?
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A prosthetic group may also be called: 1. Cofactor 2. Coenzyme
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Holoenzyme
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A complete, catalytically active enzyme together with its bound coenzyme and/or metal ions
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Apoenzyme
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Aka Apoprotein
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Apoenzyme
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The protein portion of an enzyme; protein portion of an enzyme without its prosthetic group (coenzyme or cofactor)
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Holoenzyme
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whole protein enzyme + prosthetic group
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Holoenzyme
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Complete or catalytically active enzyme
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______ act as transient carriers of specific functional groups
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Coenzymes
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Some enzyme proteins may be ________ ______.
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COVALENTLY MODIFIED. Phosphorylation Glycosylation Other processes
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What does covalently modification do to enzyme proteins?
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Covent modification of enzyme proteins causes alterations to activate/inactivate regulation of enzyme activity.
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Glycosylation
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A type of covalent modification; which is the addition of a sugar.
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Phosphorylation
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A type of covalent modification; which is the addition of a phosphate group.
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-ase suffix
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Most enzymes end in “-ase” suffix, but not all enzymes. Some enzymes were named prior to the adoption of this process.
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Exceptions to proteins ending in “-ase”
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Pepsin, trypsin, lysozyme
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-“ase”
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suffix to name enzymes; added to the name of their substrate or to a word or phase describing their activity.
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urease catalyase
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enzyme, functions for hydrolysis of urea
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DNA polymerase
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enzyme that catalyzes the polymerization of nucleotides to form DNA
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Why do not all enzymes end in “ase”?
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Some enzymes were named by their discovers for a broad function, before the specific reaction catalyzed was known.
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Pepsin
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Enzyme named not using typical “ase” suffix. Acts in digestion of foods.
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Lysozyme
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Enzyme named not using typical “ase” suffix. Named for the ability to lyse bacterial cell walls.
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Trypsin
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Enzyme named not using typical “ase” suffix. Named based on greek word tryein “to wear down”; the name was obtained based of function of rubbing pancreatic tissue with glycerin.
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EC
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Enzyme Commission
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EC system
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Enzyme commission system -divides enzymes into six classes; each with a subclass Based on the type of reaction catalyzed. -each enzyme is assigned a four-part classification number and a systematic name, which identifies the reaction it catalyzes.
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Purpose of EC system
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Enzyme commission system names an enzyme based on the class of enzyme and type of the reaction they catalyze (identify the reaction it catalyzes).
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Each enzyme has a _____-part classification number and systematic name.
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Each enzyme has a FOUR-part classification number. 6 Classes of enzymes.
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Class 1 of enzyme
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Oxidoreductases
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Class 2 of Enzyme
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Transferases
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Class 3 of enzyme
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Hydrolases
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Class 4 of enzyme
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Lyases
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Class 5 of enzyme
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Isomerases
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Class 6 of enzyme
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Ligases
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Oxidoreductases
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Class 1 enzyme
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Transferases
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Class 2 Enzyme
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Hydrolases
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Class 3 enzyme
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Lysases
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Class 4 enzyme
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Isomerases
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Class 5 enzyme
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Ligases
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Class 6 enzyme
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Reaction Type: Electron transfer (H atoms)
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Oxidoreducatase
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Reaction Type: Group Transfer
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Transferases
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Reaction Type: Hydrolysis (transfer of functional groups to water)
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Hydrolases
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Reaction Type: Formation of double bonds by removal of groups or addition to double bonds
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Lysases
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Reaction Type: Isomerizations (transfer of groups within molecules to yield isomeric forms)
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Isomerases
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Reaction Type: Condensations (formation of C-C, C-S, C-O and C-N bonds coupled to ATP cleavage)
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Ligases
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L-lactate dehydrogenase (example of EC naming)
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Catalyzes the oxidation of lactate to pyruvic acid. EC 1.1.2.3 1=oxioreductase 1=oxidation involves conversion of OH to ketone 2=cytochrome acts as electron acceptor in oxidation 3=identified L-lactate as enzymes substrate
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D-Lactate dehydrogenase
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EC 1.1.2.4. Different from L-lactate dehydrogenase, in which it uses D-lactate for the substrate of the enzyme instead of L-lactate.
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International system used to classify enzymes
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Enzyme Commisssion System (EC)
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Some enzymes are composed only of amino acids, while others may contain additional components beside the peptide chain
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The additional entity is known as a prosthetic group: -which may be organic or organometallic (coenzyme) or -cofactor (catalytic activity may require an inorganic ion)
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Cofactor
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Requires an inorganic ion for the catalytic activity of the enzyme. Often a divalent metal cation is required (Mg2+, Fe2+)
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Holoenzyme
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a complete catalytically active enzyme
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Apoenzyme
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aka apoprotein The protein part of a holoenzyme
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Sometimes a protein must undergo _____ ______ a one of its side chain functional groups in order to be catalytically active, or block catalysis
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Example of catalytic modification
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Addition of phosphate or sugar group to OH of amino acid serene.
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Enzymes
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Protein (usually) molecules that catalyze the vast majority of reactions that occur in living organisms.
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Classifications in EC
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Six different categories depending on the type of reaction catalyzed by the particular enzyme.
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Enzyme that catalyzes the oxidation of lactic acid to pyruvic acid
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lactate dehydrogenase
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In the absence of a catalyst, what would occur with most reactions?
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In the absence of a catalysts, most reactions would occur extremely slow; even though they may be thermodynamically favored.
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Without an enzyme, most reactions would occur extremely slow, even though they may be ________ ___________.
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Thermodynamically favored
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Conversion of Glucose to CO2 and Water
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Absence of catalysis= This conversion would take an extremely long time to occur under biologically relevant conditions. Presence of catalysis= This conversion would take place very fast, with a lot of intermediates in between and able to get energy from the reaction.
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What is the function of enzymes?
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Enzymes function to provide the means to vastly INCREASE the RATE of a reaction.
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Enzymes are highly _______.
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SPECIFIC So specific we need to name them in a systematic way.
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In biologically relevant conditions, do uncatalyzed reactions occur?
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Yes they occur, but are very slow. Biological molecules are quite stable in our cells (neutral pH, mild temp. and aqueous environment)
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Many common reactions in biochemistry are _____ or ______ to occur in the cellular environment.
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Unfavorable Unlikely to occur But enzymes provide a SPECIFIC environment in which a given reaction may occur more rapidly.
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Active Site
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Pocket on the enzyme where the enzyme-catalyzed reaction may take place; outside the unfavorable cellular environment.
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the molecule that is bound in the active site and acted upon by the enzyme
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substrate
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Surface of the active site…
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amino acid residues with substituent groups that bind the substrate and catalyze its chemical transformation.
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When a substrate binds to the active site….
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the active site encloses a substrate, removing it completely from solution
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Portion of enzyme in which the reaction occurs, usually a pocket or receive on the enzyme’s surface.
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the active site
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the molecule that binds to the active site and is acted upon by the enzyme
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a substrate
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amino acid residues that constitute the active site
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E S P
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Enzyme (E) Substrate (S) Product (P)
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E+S–>ES—>EP—>E + P
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A enzymatic reaction (all reversible=equilibrium reaction)
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Catalyst function to ___________. Catalyst do not affect the ______ _____.
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Increase the RATE of the REACTION REACTION EQUILIBRIUM
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____ and ____ are transient complexes of the enzyme with the substrate and with the product.
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ES and EP
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Reaction Coordinate diagram
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any reaction may be described using the energy changes during a reaction. X=reaction coordinate (time) Progress of reaction in which S is converted to P Y=free energy of system
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Ground state
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The starting point for either the forward or the reverse reaction.
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If A can’t be converted to B under any circumstance, an enzyme….
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an enzyme may never make it occur either. It must be possible for the enzyme to work.
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Activation energy
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Delta G double dagger
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The energy required to reach the transition state before the system goes on to product
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Activation energy
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Biochemical standard free energy change that occurs when S is converted to P at pH of 7.0
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biochemical standard free energy change ΔG0′
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equilibrium between S and P reflects….
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the differences in the free energies of their ground states.
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_________ reduce the activation energy required to reach the transition state.
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Enzymes
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_____ and _____ are intermediate states which occupy minima along the reaction coordinate
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ES and EP
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The ______ and ______ of equilibrium in a reaction coordinate are NOT affected by any catalyst (enzyme.
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Position Direction
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Catalyst will create a ______ activation energy of the reaction, lowering the ______ ______
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lower transition state
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In a reaction there are often distinct intermediates along the way.
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A favorable reaction doesn’t mean that the conversion of S—> will occur at a _______ rate.
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detectable
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A rate of a reaction and whether a reaction is favorable or not are TWO SEPARATE things.
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The point at which decay to the S or P state is equally probable, it is downhill either way.
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Transition State
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The _____ ______ is not a chemical species with any significant stability and should not be confused with ES or EP.
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Transition state.
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The difference between the energy levels of the ground state and the transition state
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activation energy
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The rate of a reaction reflects this activation energy; a higher activation energy corresponds to a _____ reaction, while a lower activation energy corresponds to a ______ reaction.
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Slower Faster
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How may reaction rates be increased?
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The rate of a reaction may be increased with: 1. increasing the temp. 2. catalyst
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Catalyst
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a catalyst enhances the reaction rate by lowering the activation energy.
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Function of catalyst
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lower activation energy, increase reaction rate
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The rate of the reaction will ______ by increasing the temperature.
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Increase; increasing the temp will increase the number of molecules with sufficient energy to overcome the energy barrier.
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Enzymes _____ (do/don’t) affect the reaction equilibrium.
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DON’T.
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T/F: Enzymes are used up in reactions.
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F: Enzymes are not used up in the reaction. and the equilibrium point is unaffected.
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The role of enzymes is to ________ the interconversion between S and P.
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ACCELERATE
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Is the equilibrium rate affected by an enzyme?
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No the equilibrium rate of an enzyme is NOT affected The rate of reaction is increased with an enzyme, so the reaction reaches equilibrium much faster when the appropriate enzyme is present.
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Different enzymes enhance the rate of different reactions at different levels, so much more than others.
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Enzymes generally enhance reaction rates anywhere from _____ to _____ orders of magnitude.
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5 to 17 orders of magnitude
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What are two fundamental models that have been developed to explain the substrate specificity of enzymes?
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1. Lock and Key 2. Induced Fit
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First postulated by Emil Fischer in 1954
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Lock and Key Model
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The Lock is the ____ and the key is the ____, in the lock and key model.
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Lock=enzyme Key=substrate
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Only the correctly sized key (_____) may fit into the key hole (_______) of the lock (______)
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Only the correctly sized key (substrate) may fit into the key hole (active site) of the lock (enzyme)
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Lock and key model assumes…
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a certain rigidity in the shape of the active site, a shape that is complementary to that of the substrate.
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First postulated by Daniel Koshland in 1958
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Induced-Fit model
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the enzyme’s active site has sufficient flexibility to accommodate the substitute in order to form the enzyme-substrate complex.
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induced-fit model
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this model is analogous to a glove whose shape is altered somewhat by a hand which fits into it.
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induced-fit model
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this model infers rigidity of the shape of the active site
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lock and key model
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DHFR
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dihydrofolate reductase (enzyme) -important target for the actions of certain anticancer and anti parasitic drugs.
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What are the substrates for DHFR?
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The enzyme dihydrofolate reductase (DHFR), the substrates are: 1. dihydrofolate 2. NADP+
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Each substrate binds at a specific location within the enzymes’s ______ ______ in such a way that the two molecules are able to interact.
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ACTIVE SITE In DHFR enzyme, dihydrofolate and NADP+ bind to two separate locations within the enzymes active site so the molecules are able to interact
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What is essential in order to develop drugs to effect the enzyme’s actions?
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To develop drugs to effect an enzymes action, one must know: 1. Knowledge of the active site of the enzyme 2. All molecular interactions within substrate
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Rate-limiting step
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the overall rate is determined by the step with the highest energy, when several steps occur in a reaction.
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_________ step is the highest-energy point in the interconversion S and P
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Rate limiting step
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Enzymes are very specific, readily discriminating between substrates with quite similar structures.
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Delta GB
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binding energy
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binding energy
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major source of energy used by enzymes to lower the activation energies of reactions.
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the energy derived from the enzyme substrate interactions
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binding energy
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___________ identifies the structure and function of enzymes
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X-ray crystallography
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How can enzymes be enormous and highly selective rate enhancements be explained?
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1. Covalent bonds during enzyme-catalyze reactions: substrate and enzyme functional groups (aa side chains, metal ions and coenzymes) 2. non covalent bonds between enzymes and substrate–>allows energy to lower activation energy
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the ability to distinguish between a substrate and a competing molecule
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specificity
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Once a substrate is bound to an enzyme, properly positioned catalytic functional groups aid in the cleavage and formation of bonds by a variety of mechanisms:
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1. general acid-base catalysis 2. covalent catalysis
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_____ and _____ involve transient covalent interaction with a substrate or group transfer to or from a substrate.
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1. general acid-base catalysis 2. covalent catalysis 3. Metal ion catalysis Differs from mechanisms based on binding energy (non-covalent bonds)
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proton transfers mediated by other classes of molecules
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general acid-base catalysis
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In ________ __________, a covalent bond is formed during the transition from substrate to product
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covalent catalysis
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Describe the reaction with a catalyst: A-B–H20—>A + B
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A-B +X:–>A-X + B:–H20–>A + X: + B X: nucleophilic group of an enzyme within the ACTIVE SITE A-X ( a discrete intermediate), in the end X goes back to its original form
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X:
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a nucleophilic group of an enzyme within the ACTIVE SITE
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______ is a nucleophile, which may be 1. a group in one of the side chains observed in general-acid base catalysis. 2. contained in an enzyme’s cofactor
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X:
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In covalent catalysis, hydrolysis of the bond ____ and ____ results in the formation of product B.
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A and X A-X + B:–> A + X: +B
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A metal ion pulls on the electrons of a carbonyl group, rendering carbon atoms more positive and subject to nucleophilic attack.
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Metal Ion catalysis
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REVIEW 3 types of catalysis
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Enzymatic reactions take a variety of mechanistic ways, it is important to understand when developing inhibitors.
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Enzymes: 1. 2. 3. 4.
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1. greatly enhance reaction rates by lowering activation energy without affecting equilibrium 2. form complex with substrate within the active site 3. have high degree of specificity for substrate (lock and key and induced fit) 4. multiple weak noncovalent interactions that result in lowering of binding energy, stabilizing the transition state.
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chymotrypsinogen
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an inactive zymogen from the pancreas
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zymogen
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a precursor of an active enzyme
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Many enzymes are produces in the cell as inactive precursors, or ________
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zymogens
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most enzymes utilize a combination of several catalytic strategies to bring about a rate enhancement.
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What is a good example of the use of both covalent catalysis and general acid-base catalysis?
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Reaction catalyzed by chymotrysin
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pancreatic chymotrypsin is a ______ (a type of enzyme)
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a protease, an enzyme that catalyzes the hydrolytic cleavage of peptide bonds.
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What does pancreatic chymotrypsin specifically do?
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Chymotrypsin functions as a protease specific for peptide bonds adjacent to aromatic AA residues (Typ, Phe, Tyr)
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Pancreatic chymotrypsin cleaves peptide bonds adjacent to aromatic AA residues of ___, ____ and _____.
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Typ Phe Tyr
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Chymotrypsin enhances the rate of peptide bond hydrolysis by a factor of at least _____
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10^9
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Chymotrypsin doesn’t catalyze a direct attack of water on the peptide bond….instead
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a transient covalent acyl-enzyme intermediate is forme.
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The chymotrypsin reaction has two distinct phases:
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1. acylation phase 2.deacylation phase
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in _____ phase, the peptide bond is cleaved and an ester linkage is formed between the peptide carbonyl carbon and the enzyme.
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acylation phase of chymotrypsin
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in _____ phase, the ester linkage is hydrolyzes and the nonacylated enzyme is regenerated
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deacylation phase of chymotrypsin
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What is a classic example of general acid-base catalysis and covalent catalysis?
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Chymotrypsin mechanism
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____ mechanism involves acylation and deacylation of a ____ residue
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Chymotrypsin mechanism involves acylation and deacylation of a Ser residue
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Alpha-chymotrypsin
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A protease produced by the pancreas and secreted in the small intestine.
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Chymotrypsinogen
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the inactive precursor (of alpha-chymotrypsin) produced in the pancreas
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Chymotrypsinogen is a _____ chain with _____ disulfide bonds.
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Chymotrypsinogen is a SINGLE chain with FIVE disulfide bonds.
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Formation of alpha-chymotrypsin from chymotrypsinogen results in _______________.
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Formation of the active protease involves the loss of two dipeptides (residues 14/15 and 147/148) through enzymatic cleavage.
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What are key active residue sites in alpha-chymotrypsin?
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Ser 195, His 57 and Asp 102
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The hydroxyl of _______ attacks the _____ group of the substrate.
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Ser 195 carbonyl
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What is the specific role of alpha-chymotrypsin?
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The specific role of alpha-chymotrypsin: Hydrolyzes the peptide bond in which one of the AA has an aromatic ring in its side chain (phenylalainine, tyrosine and tryptophan)
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Alpha-chymotrypsin belongs to the _____ protease family of proteins.
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SERINE
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The active site of alpha chymotrypsin…
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consists of 3 amino acids (catalytic triad): 1. histidine residue 57 2. aspartate residue 102 3. serine residue 195
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How is the three amino acids consisting of the active site of chymotrypsin (catalytic triad) are so far away from each other?
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The three residues are far apart from each other in the primary sequence of the enzyme, but the 3-D conformation of the enzyme brings them into close proximity.
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Imidazole ring of histidine (in chymotrypsin catalytic triad) simultaneously acts as ______ and ______
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proton donor proton acceptor Thus it may act as an acid or a base
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Serine hydroxyl group acts as a ________
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proton donor
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Serine ______ group acts as a proton donor to…
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Serine hydroxyl (OH) group acts a proton donor to result in an oxy anion attacks the carbonyl of the peptide bond.
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Acyl part of the peptide bond is…
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covalently attached to the serene OH group as an ester, which is subsequently hydrolyzed to regenerate the catalytic triad.
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Review mechanism of chymotrypsin slide 21
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Oldest and most important method of studying the mechanism of enzyme-catalyzed reactions.
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Enzyme kinetics
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Measurement of the rate of an enzyme-catalyzed reaction and that rate changes in response to changes in experimental conditions.
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Enzyme kinetics
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Studying the kinetic parameters of an enzyme can provide valuable insight into numerous biochemical processes, as well as the mechanisms of action for many drugs.
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Enzyme kinetics is widely used to get insight into the mechanism of which a particular enzyme works.
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Compound A converted to Product P, uncatalyzed
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Linear relationship between [A] and reaction velocity (V) The greater the concentration of A, the faster the reaction will be. As we increase the concentration of A, the reaction velocity will increase.
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1st order reaction (uncatalyzed)
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velocity of reaction (rate of reaction) is PROPORTIONAL to the concentration of the substrate [A]
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Initial rate
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aka initial velocity, Vo A measure taken when [S]>(much greater)[E] If [S] is present in five or six orders of magnitude or higher.
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Michaelis-Menten Enzyme Reaction
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E+S–>ES–>P + E Reversible reaction so equilibrium constant is K1 and K-1, then K2 and K-2
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If K2 is far larger than K-2, than
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one may say ES–>P+E is almost irreversible
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First order reaction
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a linear relationship between [substrate] and the reaction velocity, v, is established and the slope of the line is directly proportional to the [substrate], the proportionality being defined by the rate constant K
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In first order reaction, the ____ of the line is directly proportional to the [substrate], which is defined by ______.
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SLOPE The proportionality being defined by the RATE CONSTANT K
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General theory of enzyme action
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1st proposed by Leonor Michaelis and Maud Menten in 1913.
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Michaelis-Menton Model
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Based on the assumption that the enzyme (E) reversibly combined with the substrate (S) to form complex ES that breaks down to yield Product (P) and regenerate the enzyme
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In M-M model, the formation of P is irreversible
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when K-2=0
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Equilibrium constants in M-M model are
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associated with both forward and reverse reactions.
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What occurs if one plots the [S] against the velocity, V, of an enzyme-catalyzed reaction?
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A line is obtained that tends to level off at high substrate concentrations.
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The plateauing effect, that is seen at high substrate [}, occurs
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because all the enzyme’s active sites have become saturated with substrate and the reaction cannot be speed up no matter how much more substrate is added.
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When the line levels off at high [substrate],
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the enzymes active sites are saturated with substrate and the reaction rate may not increase, not matter how much more substrate is added=Vmax
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The point in the plot where a plateauing effect is seen at high [substrate] and the reaction is occurring at _________
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it’s maximum velocity, Vmax
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At Vmax, the rate of the reaction (is/isn’t) dependent on the concentration of substrate and is said to be displaying ____ order kinetics.
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At Vmax, the rate of the reaction ISN’T dependent on the concentration of substrate and is said to be displaying ZERO order kinetics.
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Michaelis-Menton Kinetics
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All reactions when plotted and the plateauing effect occurs when high [substrate] and Vmax is exhibited, are said to obey Michaelis-Menten kinetics
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Enzymes which follow Michaelis-Menton Kinetics and are plotted, the plots is referred to as….
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Saturation curve or Michaelis Menten-Plot
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Vo
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Initial velocity Measurement of velocity of the reaction immediately after the substrate and the enzyme are mixed (1 minute) at which the product [] is zero.
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Why is Vo measurement taken 1 minute after substrate and enzyme are mixed?
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Vo is taken early, while the product [] is zero and the back reaction may be ignored.
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At Vo, the [] of ______ is assumed to be constant.
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[ES] enzyme-substrate complex concentration is assumed to be constant.
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What does it mean when [ES] is constant?
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At Vo, the [ES] is constant, which means the rate at which ES forms from E and S is the same as the rate at which ES disappears both by reforming E + S and forming E+P Such a system is said to have attuned steady-state condition
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In a M-M plot, at low [substrate]…
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the formation of ES from E + S is first order; the rate of the reaction is directly proportional to the initial substrate concentration.
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The rate of the reaction is directly proportional to the initial [substrate]
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first-order kinetics which is seen at low [substrate] in M-M plot
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Km
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Michael Constant It is characteristic of each particular enzyme
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Km
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the [substrate] at 1/2 the value of Vmax, which is constant of that particular enzyme.
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Km
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value obtained by adding the rate constants K1 and K2 and dividing by K1
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(K1+K2)/K1 Where K1, K2 are rate constants of the reaction.
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Km
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1/2Vmax
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=Km
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Km= the substrate concentration at 1/2 Vmax
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Michaelis-Menten equation
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may be used to determine Vo of an enzyme catalyzed reaction at any substrate [] if one knows the values of Vmax and Km.
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Michaelis-Menton plot: Is it practical?
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The M-M plot is not practical, because there is no easy way to obtain the values of Km and Vmax. Using substrate [] sufficiently high to reach Vmax is difficult to obtain due to problems of substrate availability or cost.
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What is way to avoid the high cost of a Michaelis-Menton plot?
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Manipulation of the Michaelis-Menton Equation is developed to overcome by: Hans Lineweaver and Dean Burk (1934)
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What occurs if the reciprocal of the Michaelis-Menton equation is taken?
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If the reciprocal of the M-M equation is taken on both sides, it leads to y=1/vo (similar to y=mx + B)
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Y= X= M= B=
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M=slope of the line, Km/Vmax X=reciprocal of the substrate [], 1/[S] b=y-intercept of 1/vmax y=1/vo
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Lineweaver-Burk Plot
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A manipulation of M-M equation; which a straight-line relationship is derived when 1/Vo is plotted against 1/[S] Then Km and Vmax may be directly obtained from the double-reciprocal plot
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1/Vo is plotted against 1/[S]
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Lineweaver-Burk plot aka double reciprocal plot
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What may be obtained from a linesweaver-burk plot?
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Km and Vmax may be directly obtained from the plot.
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Vo=Vmax[S]/ ((Km +[S]))
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Michaelis-Menton Equation at any point along the graph
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KNOW Michaelis-Menten Equation
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Km (IMPORTANT DEFINITION)
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substrate [] at which an enzyme is working at 1/2 it’s maximum velocity
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y =m x + b 1/vo=km/vmax 1/[s] 1/vmax
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Reciprocal of Michaelis-Menton Equation, which produces a Linesweaver-Burke Plot
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In a Linesweaver-Burke plot, y-axis? x-axis?
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x-axis= 1/[s] y-axis=1/[Vo]
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In a linesweaver-burke plot, the y-intercept represents ___________ and the value of ____ may be obtained by extending the line to the x-intercept.
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y-intercept represent Vmax a line may be extended to x-intercept to obtain the value of Km
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The importance of Km
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may be observed from the Michaelis-Menten Plot of two enzymes that catalyze the same reaction; because each enzyme has a characteristic Km value which is constant of that particular enzyme
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______ and _______ are enzymes which catalyze the same reaction of conversion of glucose to glucose-6-phosphate (1st step in metabolism of glucose)
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Hexokinase Glucokinase Reaction that brings glucose into metabolic
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Hexokinase
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an enzyme that is primarily in the brain and skeletal muscles; it catalyzes the reaction of conversion of glucose to glucose-6-phosphate
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Glucokinase
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an enzyme that is primarily in the liver; it catalyzes the reaction of conversion of glucose to glucose-6-phosphate.
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Glucokinase and Hexokinase are examples of __________
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isozymes; enzymes that catalyze the same reaction but have different properties and are encoded by different genes
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Hexokinase has a Km of _____ mM for glucose, and its isozyme glucokinase, has a Km of ____ mM for glucose.
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0.005 mM for hexokinase 10 mM for glucokinase Km of Glucokinase is about 200 times greater than the Km of hexokinase.
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Km of Glucokinase is about 200 times greater than the Km of hexokinase. What does this indicate?
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Since the normal blood glucose concentration is 5mM, it Indicates the brain enzyme (hexokinase) may operate with great efficiency to enable the metabolism of glucose at extremely low blood glucose levels, which helps maintain brain function under dire conditions.
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Km of hexokinase would be less than Km of glucokinase, because Km is reached at very low [glucose] compared to glucokinase.
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Which enzyme be more efficient catalyst for the reaction: glucose—>glucose-6-phosphate?
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Hexokinase
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enzyme in brain; Km is very low so the enzyme is saturated with glucose at very low glucose levels.
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Amount that catalyzes formation of one micro mole of product in one minute
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one enzyme unit
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one enzyme unit
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Amount that catalyzes formation of one micro mole of product in one minute
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Number of substrate molecules converted into product per enzyme molecule per unit time when the enzyme is saturated with substrate
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turnover number (Kcat)
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Kcat
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turnover number
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Kcat
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Number of substrate molecules converted into product per enzyme molecule per unit time when the enzyme is saturated with substrate
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An enzyme at saturation, Kcat, is how many molecules are converted to product
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Kcat of a particular enzyme is a ______
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Kcat, turnover number, of a particular enzyme is CONSTANT
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[Vmax]/total amount of enzyme in=
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Kcat (sec-1) expresses in units of reciprocal seconds
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Kcat values of enzymes
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vary; a wide distribution.
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Kcat describes….
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the limiting rate of any enzyme-catalyzed reaction at saturation. If a reaction has several steps and one is clearly the rate limiting step, Kcat is equivalent to the rate constant for that limiting step.
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A large Kcat value indicates _______, while a small Kcat value indicates _________.
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Kcat allows us to compare the relatives velocities of catalysis of various enzymes, but doesn’t tell us anything about the enzyme’s affinity for its substrate. Km tells us about affinity, but nothing about velocity.
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What is enzyme activity affected by?
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1. Temperature 2. pH 3. Covalent modifications 4. Presence of inhibitors
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How does pH affect enzyme activity?
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Each enzyme is optically active at a certain pH, which is generally near the pH of the medium in which the enzyme operates.
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pH of the medium may be important in determining the efficiency of the enzyme
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The enzyme has a maximum efficiency at the pH of which it is found (in this case, the tissue in which it is found)
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Pepsin
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enzyme that has maximum capacity at pH of 1-2; energineered to be maximum effective at a low pH
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pH has an affect on an enzyme because the side chains may be ionized or unionized depending on the pH
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Salivary amylase
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enzyme found in the mouth that has the maximum capacity at pH of 5-7
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Alkaline phosphatase
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enzyme found in bone and tissue and has the maximum capacity at pH of 9
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Enzyme has an optimum pH (or pH range) at which their activity is maximal; at higher or lower pH, the activity decreases.
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Temperature on enzyme activity
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For most enzymes, we find maximum enzyme activity at/around body temp, or the environment they function in. If you increase temp. above body temperature, the enzyme is denatured and the active site will be affected.
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Thermophilic organisms
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Thermus aquatics are organisms that thrive at high temperatures and possess enzymes that operate efficiently at high temps. Organism is the source of Taq DNA polymerse
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Taq DNA polymerase
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Important enzymes in molecular biology due to it’s use in PCR (polymerase Chain reaction) Organism Thermus aquatics is the source for Taq DNA polymerase
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Most enzymes are subject to _____ _____, which may greatly influence their activity.
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Covalent modifications May be used to control an enzyme activity
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Kinase
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aka phosphorylation enzyme that phosphorylates a substrate to activate or deactivate.
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phosphatase
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an enzyme that removes a phosphate to activate or deactivate an enzyme.
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Phosphorylation occurs especially on ____ groups, like _____, ______ and ______
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Phosphorylation occurs especially on the OH groups, like Threonine, Tyrosine, and Serine; which may affect the activity of an enzyme.
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acetyl CoA carboxylase
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an enzyme involved in the production of FA in the cell; the enzyme may be inactivated by phosphorylation of a specific OH group by a particular type of kinase.
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Acetyl CoA carboxylase by be (activated/inactivated) by phosphorylation by a kinase, and (activated/inactivated) by dephospho rylation by a phosphatase
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INACTIVATED by a kinase (addition of a phosphate group) ACTIVATED by a phosphatase (removal of a phosphate)
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What are the various types of enzyme inhibition?
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1. Reversible -Competitive -Uncompetitive (uncommon/don’t need to know) -Noncompetitive *Pure (uncommon) *Mixed (most noncompetitive enzyme inhibitors are mixed) 2. Irreversible (DISTINCT COVALENT BOND FORMATIO -Covalent bond formation -Suicide (mechanism-based) inactivators
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What is the major difference between reversible and irreversible enzyme inhibition?
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Irreversible enzyme inhibition involves COVALENT BOND FORMATION, while reversible enzyme inhibition doesn’t.
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Suicide enzyme inactivator
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Special type of irreversible inhibitor in which the inhibitor is converted by the enzyme to an activated form, capable of forming a covalent bond within the active site.
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Irreversible inhibitors
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Enzymes that bind covalently with or destroy a functional group on an enzyme that is essential for the enzyme’s activity or those that form a particularly stable non covalent association.
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In irreversible inhibition, ______________ is common.
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formation of a covalent link between an irreversible inhibitor and an enzyme are common.
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Amino acids with key catalytic functions in the active site can sometimes be identified by determining which residue is covalently linked to an inhibitor after the enzyme is inactivated.
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A special class of irreversible inhibitors
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suicide inactivators.
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What is one type of reversible inhibitor?
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A type of reversible inhibitor is COMPETITIVE inhibitor.
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Competitive Inhibition
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A competitive inhibitor competes with the substrate for the active sit of an enzyme. When the inhibitor (I) occupies the active site, it prevents the binding of the substrate to the enzyme.
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Many competitive inhibitors are compounds that resemble _______
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resemble the substrate and combine with the enzyme to form the EI complex, but without leading to catalysis.
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The inhibitor (of competitive inhibition) often bears a structural resemblance to the substrate; however, the complex (EI) formed between enzyme and inhibitor doesn’t lead to catalysis.
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A competitive inhibitor (I) binds to the enzyme’s active site with it’s own equilibrium constant (K1), preventing….
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the normal substrate from binding to the site.
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Because inhibitor binds reversibly to the enzyme, the competition….
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can be biased to favor the substrate, by adding more substrate. When [S] far exceeds [I], the probability that an inhibitor molecule will bind to the enzyme is minimized and the reaction exhibits a normal Vmax.
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How does competitive inhibition affect the M-M equation?
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In competitive inhibition, a normal Vmax may be seen when [S] far exceed [I]. BUT, the [S] at which Vo=1/2Vmax, the Km value will increases in the presence of an inhibitor by a factor of alpha.
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In this type of inhibition, the Vmax remains normal, but the Km value will increase by a factor of alpha.
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Competitive Inhibition.
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Vo=Vmax[S]/ (((alphaKm) + [S]))
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competitive inhibitor in the system; therefore, Km is increased by a specific amount (alpha)
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K1=[E][I]/[E]
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Rate constant of a competitive inhibitor reaction
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In a Michaelis-Menten plot, [S] (x-axis) is plotted against Vo for a competitive and noncompetitive inhibitor….
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Competitive inhibitor: -Vmax doesn’t change compared to noncompetitive -Km, substrate concentration required to reach 1/2Vmax is increased in presence of competitive inhibitor Noncompetitive inhibitor:
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the substrate concentration required to reach 1/2Vmax
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definition of Km
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In the presence of a competitive inhibitor, compared to a noncompetitive inhibitor:
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1. The Vmax remains the same 2. Km is increased in the presence of a competitive inhibitor.
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Why does Vmax remain the same in the presence of a competitive inhibitor, compared to a noncompetitive inhibitor?
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A Vmax doesn’t change, because increase the [substrate] overwhelms the effect of the inhibitor so the maximum velocity of the reaction may still be achieved.
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Why does Km change in the presence of a competitive inhibitor?
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The Km changes in the presence of a competitive inhibitor, because the substrate [] required to reach 1/2 vmax (aka Km) is increased in the presence of a competitive inhibitor.
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In a competitive inhibitor, describe a typical lineweaver-burke plot.
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Competitive Inhibition: Lineweaver-Burke Plot 1. All have same y-intercept (1/Vmax); because the Vmax remains the same. 2. Km (x-intercept) is increased by a factor of the reciprocal of alpha (1/Km*alpha)
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In a competitive inhibitor, describe a typical lineweaver-burke plot:
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Add more inhibitor, the slope line is increased and a steeper line occurs. Vmax is the same Km is increased
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alpha=1 + [I]/K1
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How does competitive inhibition affect the Km?
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What is a classic example of competitive inhibition?
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A classic example of competitive inhibition is: Succinate Dehydrogenase
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Succinate Dehydrogenase
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enzyme involved in step 8 of the citric acid cycle
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Competitive inhibitor of succinate dehydrogenase
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Malonate
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Malonate
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competitive inhibitor of succinate dehydrogenase
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malonate
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bears an obvious structural resemblance to succinate substrate and is capable of binding reversibly to enzyme’s active site.
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Substrate, ______, and competitive inhibitor __________ compete to bind to the active site of the enzyme succinate dehydrogenase.
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Substate-succinate CI-Malonate
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Malonate is an inhibitor of cellular respiration, because it binds to the active site of the succinate dehydrogenase in the citric acid cycle but does not react, thereby competing with succinate. For the oxidative phosphorylation reaction, Malonate is an inhibitor for complex II which, again, contains succinate dehydrogenase.
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Succinate Dehydrogenase (E) + Succinate (S)–>ES complex—> Fumarate (P) and Succinate dehydrogenase (E)
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In competitive inhibition, Succinate Dehydrogenase (E) + Malonate (S)–>ES complex—> No product is formed, nothing occurs malonate just physically takes up space in the active site.
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ADH
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Alcohol dehydrogenase
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Example of an enzyme subject to competitive inhibition
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Alcohol Dehydrogenase (ADH)
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Methanol poising
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Liver enzyme converts methanol to formaldehyde (damaging to many tissues); blindness is often a result of methanol ingestion. Eyes are sensitive to formaldehyde.
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ADH
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Ethanol and Methanol compete to occupy the active site of ADH (alcohol dehydrogenase)
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Ethanol–ADH–>acetylaldehyde Methanol–ADH–>formaldehyde–>formic acif
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Ethanol competes much like a competitive inhibitor for ADH.
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Methanol Poising: Treatment
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In methanol poising, methanol is converted in the body by ADH enzyme to formaldehyde. Formaldehyde is a toxic substance, which the eyes are extremely sensitive to and may cause blindness. Use ethanol to displace the methanol from the active site of ADH enzyme, by giving a slow IV infusion of ethanol at a rate that maintains a controlled concentration in the bloodstream for many hours.
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Result of Methanol Poising Treatment
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Result: Slows the formation of formaldehyde, lessing the danger while the kidneys filter out the methanol to be excreted harmlessly in the urine.
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The enzyme alcohol dehydrogenase is not specific as to which substrate it binds to; 1. ethanol or 2. methanol
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Acetylcholine
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know structure
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a neurotransmitter widely found in the ANS and CNS
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acetylcholine
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What are the neurotransmitter affects of acetylcholine?
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1.Stimulates skeletal muscle contractions 2. Inhibits cardiac muscle contractions 3. Stimulate GI secretions 4. Role in cognitive function (closely associated with alztimers)
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Acetylcholinesterase
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aka cholinesterase, an enzyme
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classic example of a enzyme that frequently has been the target in the development of drugs, by competitive inhibition
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acetylcholinesterase, aka cholinesterase.
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substrate for acetylcholinesterase
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acetylcholine
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the role of acetylcholine in cognition has….
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led to the development of cholinesterase inhibitors in the treatment of Alzheimer’s disease.
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ACh
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Acetylcholine
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ACh
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a neurotransmitter in cholinergic neurons ACh is released by the pre-synaptic neuron and after interacting with the acetylcholine receptor on the post-synaptic neuron, its action is terminated by acetylcholinesterase.
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ACh
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is cleaved by acetylcholinesterase on the post-synaptic neuron ACh receptor, into acetate and choline, which are then taken up by the pre-synaptic neuron for synthesis of more acetylcholine
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Mechanism by which Acetylcholinesterase hydrolyzes acetylcholine involves:
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3 residues in the active site.
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What are the 3 residues in the active site of acetylcholinesterase responsible of hydrolyzing acetylcholine?
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3 residues in active site: 1. Glu 334 2. Ser 203 3. His 447
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+charged quaternary nitrogen atom of ACh is held in place by __________
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gluatamate residue (334) anionic site
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All while the serine OH group attacks the ________ with the assistance of __________
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All while the serine OH group attacks the ESTER CARBONYL with the assistance of the IMIDAZOLE RING of the histidine residue (esteratic site)
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The acetylated intermediate is then..
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hydrolyzed. Then the vacated anionic and esteratic sites are then free to accommodate another molecule of acetylcholine.
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ACh has a high Kcat value
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A classic example of a cholinesterase inhibitor
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neostigmine
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neostigmine
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a drug, cholinesterase inhibitor, which blocks the breakdown of acetylcholine in the synapse (synaptic cleft)
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What is the result of neostigmine?
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Neostigmine, a cholinesterase inhibitor, blocks the breakdown of acetylcholine in the synapse—>cholinergic agent.
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Cholinergic agent
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a drug that has effects are similar to that of ACh itself.
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Why is neostigmine differ from ACh?
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Neostigmine occupies the same active site as ACh, and its carbonyl group (carbonate group) is subject to attack by the serine OH, just like ACh. BUT the intermediate carbamate ester is hydrolyzed MUCH MORE SLOWLY than is an ACETYL ESTER–>thus effectively blocking the active site.
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Neostigmine occupies the same active site as acetylcholine and its carbonyl group is subject to attack by serine OH, just like ACh….but
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the intermediate carbamate ester of neostigmine is hydrolyzed MUCH MORE SLOWLY than is an acetyl ester–>thus effectively blocking the active site.
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Trial by Ordeal
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Persons accused of crimes or witchcraft were given a Calabar bean to chew (which contains physostigmine). If death resulted (usually by respiratory depression), the person was deemed to be justifiably guilty. However, if the person’s stomach rejected the agent, they were said to be proven innocent. To avoid death, one must vomit the Calabar bean up.
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Physostigmine
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a naturally occuring reversible cholinesterase inhibitor with cholinergic effects; especially useful in treating glaucoma.
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Found in Calabar bean (physostigma venenosum) a plant native to Nigeria
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Physostigmine, a naturally occuring reversible cholinesterase
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used to treat glaucoma
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physostigmine (found in Calabar bean)
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Bind to both the Enzyme (E) and the Enzyme-Substrate (ES) complex
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Noncompetitive inhibitors
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In noncompetitive inhibition, the inhibitors (do/don’t) bind at the same spot as the substrate, so the inhibitors effect (can/can’t) be overcome by increasing [S]
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DON’T CAN’T
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In ______ inhibition, binding of the inhibitor to the substrate INFLUENCES the binding of the substrate to the enzyme.
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NONCOMPETITIVE
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Where are the binding sites in noncompetitive inhibition?
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In noncompetitive inhibition, the binding sites for inhibitor and substrate are separate on the enzyme, they may be near each other or the inhibitor may induce conformational changes in the enzyme that affect binding of the substrate to the enzyme.
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In noncompetitive inhibition, the competitor in effect lowers the ____________
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concentration of the enzyme. Because an inhibitor may induce conformational changes in the enzyme, which affect binding of the substrate to the enzyme.
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In noncompetitive inhibition, the Vmax is _____ and the Km is _____ in the presence of a noncompetitive inhibitor.
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Vmax is REDUCED Km is INCREASED
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What are the types of noncompetitive inhibition?
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Two types of noncompetitive inhibition: 1. pure 2. mixed
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Pure none competitive inhibition
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uncommon occurs when inhibitor binds to the E and ES with the same affinity (K1=K1′)
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Mixed competitive inhibition
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frequently occurs substrate and the inhibitor have different affinities for their respective binding sites K1 doesn’t equal K1′ (like pure noncompetitive)
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Lineweaver-Burke plot of pure noncompetitive inhibitor
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Km is not affected by the inhibitor Vmax decreases
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Lineweaver-Burke plot of mixed noncompetitive inhibitor
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Km effect depends Vmax decreases with increased concentrations of inhibitor.
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Irreversible Inhibition
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similar to reversible inhibition, except in irreversible inhibition the inhibitor forms a covalent bond with the enzyme, which is indicated by *. EI*
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Differentiate between reversible and irreversible inhibitors.
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Reversible inhibitors: non covalent bonds formed Irreversible inhibitors: covalent bonds formed with an enzyme and inhibitor, which are permeant bond to make the interaction irreversible!
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On a lineweaver-burke plot, distinguish competitive and noncompetitive inhibition.
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Competitive inhibition: 1. Vmax is unchanged (y-intercept) 2. Km increases (x-intercept) 3. effect of inhibitor may be overcome by increasing [substrate] Noncompetitive: 1. Vmax decreases with increased concentration of inhibitor 2. Km increases 3. effect of inhibitor may not be overcome by increasing [substrate]
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Examples of irreversible inhibition
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acetylcholinesterase enzyme: Irreversible inhibitors: phosphonate esters containing a good leaving group.
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Phosphonate esters
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a irreversible inhibitor of acetylcholinesterase. Phosphonate ester is formed at serine OH group, such esters are extremely difficult to hydrolyze–>the enzyme’s active site is irreversibly blocked.
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The effect of an irreversible inhibitor can’t be overcome by the addition of more substrate, just like _____ inhibitor
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noncompetitive reversible inhibitor.