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43 Cards in this Set
- Front
- Back
Which are affected by the catalytic activity of enzymes?
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a) Transition state
b) Rate constant for the reaction c) Standard free energy of activation d) Rate at which the reaction reaches equilibrium |
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Describe catalytic mechanism triose phosphate isomerase...
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a) Acid-base catalysis: H+ ions are transferred between enzyme and substrate
b) H+ is pushed in addition to electron pairs c) Not a redox reaction – No covalent bonds are formed d) No hydrolysis occurs |
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Catalytic mechanism for Chymotrypsin?
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a) Protease – catalyzes hydrolytic cleavage of peptide bonds
b) Uses covalent catalysis, general acid-base catalysis, and transition-state stabilization |
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Two compounds pyruvate could be converted into under anaerobic conditions (excluding cellular resp.)
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a) Lactate fermentation
i) Lactate dehydrogenase – enzyme ii) NADH + H NAD+ b) Alcohol fermentation i) Acetaldehyde then ethanol ii) Pyruvate decarboxylase (releasees CO2) and irreversible iii) Alcohol dehydrogenase – NADH + H NAD+ |
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How many substrate-level phosphorylation steps does glycolysis have?
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2
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Substrate-level phosphorylation...
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Formation of ATP by phosphoryl group transfer from a substrate.
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How many C in glucose?
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6
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How many C in pyruvate?
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6
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How many pyruvate molecules produced by a single glucose?
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2
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“Gateway” step of glycolysis?...the "Ike" of Glycolysis?
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a) Step 3
b) Fructose 6-phosphate<->Fructose 1,6-bisphosphate c) Phosphofructokinase is the gateway!!!!! |
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Describe Phosphofructose kinase-1 mechanism...
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*highly allosteric regulated enzyme - activity increased when cell’s ATP supply is depleted or when ATP breakdown products, ADP and AMP accumulate – enzyme is inhibited whenever the cell has ample ATP
*an allosteric enzyme made of 4 subunits and controlled by many activators and inhibitors. *catalyzes the important "committed" step of glycolysis, the conversion of fructose 6-phosphate and ATP to fructose 1,6-bisphosphate and ADP. |
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Linewaver-Burke plot
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*Reciprocal of Michaelis-Menten used to plot Lineweaver-Burk plot
i) X-axis – 1/[S] ii) Y-axis 1. Competitive 1/V0 = Vmax[S]/alphaKm + [S] 2. Uncompetitive 1/V0 = (Km/Vmax) 1/[S] + alp’/Vmax 3. Mixed inhibition 1/V0 = (alpKm/Vmax) 1/[S] + alp’/Vmax |
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Competitive inhibitor
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Competes with substrate for the actie site of an enzyme, forms EI complex which does not lead to catalysis, because it is reversible – competition can be biased...
i) High [S] – normal Vmax ii) [S] which V0 = ½ Vmax increases in the presence of inhibitor by factor of alpha |
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Uncompetitive inhibitor
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Binds at a site distinct from the substrate active site and binds only to ES complex
i) High [S], V0 approaches Vmax/alp’[S] – lowers measured Vmax and apparent Km |
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Enzyme inhibitors can bind...?
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Reversibly or Irreversibly
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Allosteric enzymes function through...
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REVERSIBLE noncovalent bonding of regulatory compounds called allosteric modulators/effectors
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Enzyme inhibitors always act to reduce the velocity of the reaction catalyzed by the enzyme but in different ways...
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either competing with the enzyme to form EI complex or changing the shape of the substrate
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Allosteric effects DON’T always...
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reduce velocity of reaction, homotropic effectors act to increase the rate by creating a better fit after conformational change
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Heterotrophic allosteric effectors bind to a separate...
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“regulatory” subunit which may have a positive or negative effect on the enzyme – so not all allosteric effectors bind on the active site
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Allosteric enzyme plots...
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*produce a sigmoid saturation curve rather than hyperbolic curve of typical non regulatory enzymes
*K0.5 and [S]0.5 used instead of Km |
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Homotropic allosteric enzymes generally are...
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Homotropic allosteric enzymes generally are multi- subunit proteins and the same binding site on each subunit functions as both the active site and the regulatory site.
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In Homotropic allosteric enzymes the substrate most commonly acts as...
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*Most commonly, the substrate acts as a positive modulator (an activator), because the subunits act cooperatively: the binding of one molecule of substrate to one binding site alters the enzyme’s conformation and enhances the binding of subsequent substrate molecules.
*This accounts for the sigmoid rather than hyperbolic change in V0 with increasing [S]. In Sigmoid kinetics, a small change in [M] can be associated with large changes in activity |
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Heterotropic Allosteric enzymes...
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Different responses to their substrate-activity curve because some have negative inhibitor modulators (more sigmoid substrate-saturaion curve with increase in K0.5) (b, -), positive activating modulators (may cause curve to be more hyperbolic with decrease in K0.5 but no change to Vmax) (b, +), and some may have both! --- Graphs: (b) central curve shows the substrate-activity relationship without modulator. (c) A less common modulation, in which Vmax is altered and K0.5 is nearly constant
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Noncompetitive Inhibitors bind to...
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a siter outside the active site, destroys some enzymes so it decreases Vmax but does not change Km
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Feedback inhibition is an example of...
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enzyme regulation by an allosteric effector that turns off enzyme activity
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Michaelis-Menton equation...3 assumptions
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1. V0 – no product has been formed yet so there’s no k-2¬ reaction
2. The conversion of ES to E and product is one step a) k¬cat is rate-determining slowest step 3. Steady State assumption a) Rate forward (k1) equals the rate backward (k-1) |
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The lower the value of Km...
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The lower the value of Km, the more efficient the enzyme is with that substrate
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The larger the k1, the smaller the k-1, the...
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The larger the k1, the smaller the k-1, the better the substrate and enzyme come together
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The higher the Vmax the...
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The higher the Vmax the better the substate
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Lower kd means...
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Lower kd means higher binding affinity
(k1/k-1) |
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Oxidation state for C atom
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a) Carbon loses more electrons to its neighbors – oxidation increases
b) More Oxygen, more oxidation |
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Reaction rate
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V = k1 [A1] [A2]
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Chymotrypsin mechanism Step 1
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*Substrate binds to chymotrypsin with side chain of residue adjacent to peptide bond to be cleaved in a hydrophobic pocket
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Chymotrypsin mechanism Step 2
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*Ser195 attacks the peptide carbonyl group, breaking the double bond and forming a short-lived negative charge on carbonyl by pulling H+ off Ser
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Chymotrypsin mechanism Step 3
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*Instability of negative chg leads to collapse of tetrahedral intermediate; reforming the double bond with C, displacing C-NH bond – breaks peptide bond and AMINO group leaves and is protonated by HIS57
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Chymotrypsin mechanism Step 4
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*incoming water molecule is deprotonated (H+ gets pulled off_ by general-acid catalysis, attacks hydroxide on the ester linkage of the acyl-enzyme which generates a second tetrahedral intermediate with oxygen in the oxyanion hole w/ neg. charge
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Chymotrypsin mechanism Step 5
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*collapse of tetrahedral intermediat forms second product – a carboxylate anion and displaces Ser195
*Diffusion of second product from active site regenerates free enzyme |
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Chymotrypsin Mech Step 1-5 Summarized
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Substrate binds -> Induced fit -> Asp 102 – His 57 Hydrogen bond is compressed -> His 57 deprotonates Ser 195, making it a better nucleophile -> Ser 195 attacks carbonyl -> tetrahedral intermediate -> acyl enzyme intermediate -> His 57 depronates water -> water attacks -> tetrahedral intermediate -> products.
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What if serine was substituted with the nonpolar alanine?
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Because alanine is nonpolar a site-directed mutagenesis on chymotrypsin so that serine is ssubstituted with alanine would affect the 2nd step, alanine would not be able to attack the peptide carbonyl bond. H+ would not be pulled of alanine, and this would halt the rest of chymotrypsin mechanism
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Steady state assumptions equation
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a) k1([Et] – [ES]) x [S] = k-1[ES] + k2[ES]
b) in terms of reaction velocities |
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Glycolysis coversion of glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate, and then to 3-phosphoglycerate...
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insert pic or write out mechanism / reaction description...
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Types of Reactions
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a) Oxidoreductase – transfer of electrons (redox), includes dehydrogenase
b) Transferases – move chemical groups between two different substrates (includes kinases), always 2nd order reactions with atleast two substrates c) Hydrolases – hydrolysis “cut with water” (sever covalent bonds, etc) d) Lyases – form or remove carbon-carbon bonds by addition/removal of chemical group e) Isomerases – move chemical groups inside one substrate f) Ligases – form single bonds between a variety of different atoms; C-C, C-O, C-S, C-N (condensation reactions) – ATP provides energy for bond formation (DNA ligase is an example) |
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Classes of amino acids
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*Nonpolar aliphatic R groups (7)
Alanine, Guanine, Isoleucine, Leucine, Methione, Proline, Valine *Aromatic nonpolar R groups (3) Phenylalanine, Tyrosine,Tryptophan *Polar Uncharged R groups (5) Aspagine, Cysteine,Glutamine, Serine, Threonine *Positively charged Basic R groups(3) Histidine, Lysine, Arginine *Negatively charged Acidic R groups (2) Asparatate & Glutamate |