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31 Cards in this Set
- Front
- Back
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What is enzyme inhibition used for?
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Used physiologically for the control of metabolism, both locally and on a larger scale Inhibitors determine whether an enzyme activity mechanism is specific |
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Two types of enzyme inhibition
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Irreversible - you cannot remove the inhibitor from the enzyme Reversible - under correct conditions the inhibitor can be released from the enzyme |
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Irreversible inhibition (drug industry)
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Inhibitors bind tightly to the enzyme and react with a group at the active site of the enzyme, forming a strong covalent bond which cannot be removed. Very specific - e.g. aspirin or penicillin |
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How does aspirin work?
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Inhibits cyclooxygenase activity (COX 1 and COX 2) of prostaglandin H synthase irreversibly which prevents conversion of Arachidonate to prostaglandin H2. Forms a covalent bond with a serine 530 residue in active site by removing an acetyl group and attaching to the hydroxyl group. Substrates can no longer bind to the active site. |
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How does penicillin work?
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Covalently modifies the enzyme transpeptidase by forming a covalent bond with the hydroxyl residue so it can't easily be removed. Hydroxyl residue in Penicillin binds to the active serine hydroxyl enzyme which prevents the bacteria from existing. |
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Reversible competitive inhibition
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Bind to the same site on the enzyme as the substrate (similar structure), the active site. Competes with substrate for binding . As substrate concentration increases then competition decreases as less inhibition occurs Concentration dependent |
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Reversible non competitive inhibitors
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Do not bind to active site, bind to alternative part of enzyme. This alters shape of protein so active site no longer favourable for substrate to bind. Competition does not occur |
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The use of competitive inhibition for cancer therapy
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Methotrexate (inhibitor) inhibits dihydrofolate reductase (enzyme) by competing with dihydrofolate (substrate) for binding. Methotrexate binds more strongly than usual substrate, so dihydrofolate cannot bind and DNA synthesis is blocked so cancer cells cannot grow in S phase. Diydrofolate used for synthesis of purines and pyrimidines for DNA and RNA synthesis |
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Use of competitive inhibition to control metabolism SUCCINATE DEHYDROGENASE |
Catalyses oxidation of succinate to fumarate using FAD Malonate competitive inhibitor of succinate as it has a similar structure, they both compete for binding on succinate dehydrogenase. If malonate binds no reaction occurs so TCA cycle halted |
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Use of competitive inhibition to control metabolism FUMARASE |
Catalyses hydration of fumarate to malate. Succinate competitive inhibitor of fumarate, lacks double bond so no hydration reaction occurs when it binds. If succinate builds up it will bind to fumarase so the reaction will slow |
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How competitive inhibition works
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An enzyme inhibitor complex forms which will not produce a product so the reaction does not proceed. Mutually exclusive binding of substrate and inhibitor, only one or the other can bind. If substrate concentration increases then inhibition will be overcome |
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Curve produced with competitive inhibition
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Hyperbolic curve - rate increases with increasing substrate concentration until it becomes a limiting factor and plateaus. With increasing inhibitor - rate of reaction reduced as some enzyme bound to inhibitor. THe more inhibitor the more reaction is reduced |
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Effect of competitive inhibition on Vmax and Km
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With increasing substrate concentration you can overcome inhibition and meet same Vmax over a long time. Km increases as inhibitor concentration increases Lineweaver-Burk plot produces straight line graph, With inhibition the y intercept will be the same but x intercept different has Km increased. |
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How non competitive inhibition works
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Enzyme inhibitor substrate complex formed which stops reaction occurring as reaction blocked. Substrate and enzyme can bind in any order Inactive complex so cannot overcome inhibition by adding more substrate. |
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Graph of non competitive inhibition
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No inhibitor - rate of reaction increases to point where Vmax is reached Inhibitor - rate of reaction decreased, Vmax decreased but Km unchanged (concentration at half maximal velocity is same independent of inhibitor) Lineweaver Burk plot - different y intercept, but same x intercept as non inhibition line |
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How to distinguish between competitive and non competitive inhibition using the Lineweaver Burk plot
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Competitive - intersect on y axis and not x Non competitive - intersect on x axis but not y |
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Mixed inhibition
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Non competitive but both Km and Vmax are changed Inhibitor binds differently to enzyme on its own compared to when substrate has bound, inhibitor prefers to bind before substrate. If high concentration of substrate, inhibitor finds it difficult to bind |
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Uncompetitive inhibition
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Where inhibitor only binds to ESC but not enzyme alone Products not produced, as enzyme binds after substrate. Cannot be overcome by increased substrate concentration Vmax and Km reduced |
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Define Sequential reactions
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A and B bind together on enzyme to form ternary complex EAB in equilibrium: A + B ⇄ P + Q |
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Define Double displacement reactions
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No ternary complex is formed, both A and B do not need to be bound before release of products
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Common sequential reactions
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Used by dehydrogenase enzymes Lactate dehydrogenase reduces pyruvate to lactate, producing NAD. NADH binds first, then pyruvate. Lactate released first followed by NAD. Ternary complex formed before release of products so substrate binding is orderd. Random binding occurs in creatine kinase (phosphorylation) |
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Reactions in double displacement reactions
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Enzyme binds to substrate A which modifies enzyme and product P released B modifies enzyme and product Q released (E' where enzyme temporarily modified) E + A ⇄ E’ + P then E’ + B ⇄ E + Q A causes this which allows B to bind to modified enzyme |
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Example of a double displacement reaction
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Nitrogen metabolism - asparatate aminotransferase catalyses transfer of amino group from aspartate onto alpha-ketoglutarate to produce glutamate. Oxaloacetate is released.
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The mechanisms of enzyme catalysis
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Rate enhancement over corresponding chemical reactions - enable kinetically unfavourable reactions to occur (reduces activation energy and stabilises them to a transition state). Highly specific |
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Mechanisms by which enzymes are so efficient THEY ARE CATALYSTS |
They accelerate progress to equilibrium, but don't affect position of equilibirium Reduce activation energy by stabilising the transition state - it is entropically unfavourable for two reactants to be brought together. When substrate binds to enzyme the charges are neutralised. |
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mechanisms by which enzymes are so efficient ACTIVE SITE IS CLEFT IN THE ENZYME |
Substrate fits by shape and charge and is held in appropriate orientation for neutralising of charges. Substrate binds non covalently (H2, electrostatic) so less energy invested as weak interactions |
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Mechanisms by which enzymes are so efficient INDUCED FIT MODEL |
Substrate binds by induced fit, allowing binding of transition state to help stabilise it further and lower activation energy. Accelerates reaction and provides specificity.
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What is chymotrypsin?
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Proteolytic enzyme used in digestion. Highly specific of peptide bonds it cleaves during hydration reaction - only at carboxyl side of large hydrophobic aromatic amino acids e.g. alanine. |
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Non covalent catalysis by chymotrypsin
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Serine 195 (195th amino acid in amino acid sequence, found at active site) hydrogen bonded to histamine 57 and aspartic acid 102. Increases reactivity of serine as alkoxide ion produced which is highly reactive |
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Covalent catalysis
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once peptide bonds hydrolysed, acylation occurs which produces an acyl enzyme intermediate by nucleophilic attack from highly reactive serine. This takes part in a hydration reaction where deacylation occurs, cleaved peptide released and enzyme recycled. Reaction would not occur without serine residue |
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The specificity of chymotrypsin
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Only cleaves peptide bonds of large hydrophobic amino acids Active site has 3D shape for binding, pocket is hydrophobic. Peptide can only bind in one specific way and peptide is epcific for that amino acid |
