Biochemistry lecture 7

Michaelis - Menten equation

Used to describe

• 𝑣 is rate of velocity at a specified [S]​

• Vmax is maximum value of v attainable by the enzyme under the given conditions (impossible to reach in practise but can be estimated)​

• [S] substrate concentration​

• Km = Michaelis constant (see previous lecture)​

Michaelis kinetics

Curve indicates saturation of enzymes with substrates

Kcat

• way of comparing enzymes 

• the turnover number, is a useful measure of enzyme efficiency​

• Maximum number of molecules of substrate converted per active site per time (usually s), how many they can turnover in certain amount of time​

• Kcat = Vmax/[Et]​​

• E.g. chymotrypsin = 100 s-1​

What type of constant is Kcat?

• is the specificity constant, useful if you wish to compare the activity of an enzyme against different substrates. Units of s-1 M-1​

• Higher is better!​

Preferred substrate of aldehyde dehydrogenase

BP1 - its classification

BP2 - reactions

Calc Kcat/Km can measure enzyme with highest enzyme activity - useful to compare enzyme with varying substrates or if activity changes in various conditions

How is enzyme activity controlled?

• Enzyme inhibitors​ - Reduce the rate of enzyme catalysed reactions​

• Classified based on how they act:​

• Irreversible - usually bind to AS uncontrollably via C bond​

• Reversible:​

1. Competitive​

2. Non-competitive - binds to allosteric site​

3. uncompetitive - bind after S binds to lock it in​

Irreversible inhibitors

• Bind irreversibly to enzyme​

• Usually bind via a covalent bond ​

• Bind to an amino acid side chain at or near the active site ​

• Commonly bind to either Ser (-CH2-OH) or Cys (-CH2-SH) side chains ​

• Binding permanently inactivates the enzyme​

• Usually prevents substrate binding

Sarin (example)

• Di-isopropylfluorophosphate:​

• Sarin nerve gas​

• Covalently binds to a serine residue in acetylcholine esterase​

• Prevents breakdown of the neurotransmitter acetylcholine​

• Leads to continual activation of nerves in muscles

Aspirin 

• Salicylic acid a component of willow bark​

• Acetylated form produced in late 19th Century by Bayer​

• Key medicine with uses as an anti-inflammatory drug​

• Binds to Prostaglandin H2 synthase at S530 and transfers acetyl group​

Penicillin (I inhibitor)

• Binds to Serine in PBPs​

• Irreversibly blocks active site​

More examples

• Aspirin, which inhibits cyclooxygenase 1 and 2 enzymes.​

• Penicillin, which inhibits DD-transpeptidase from building bacterial cell walls.​

• Sulbactam, which prohibits penicillin-resistant strains of bacteria from metabolizing penicillin.​

• Allopurinol, which inhibits uric acid production by xanthine oxidase in the treatment of gout.​

• AZT (zidovudine) and other chain-terminating nucleoside analogues used to inhibit HIV-1 reverse transcriptase in the treatment of HIV/AIDS.​

• Eflornithine, one of the drugs used to treat sleeping sickness, is a suicide inhibitor of ornithine decarboxylase.​

• Sarin is a suicide inhibitor of acetylcholinesterase.​

• 5-fluorouracil acts as a suicide inhibitor of thymidylate synthase during the synthesis of thymine from uridine. This reaction is crucial for the proliferation of cells, particularly those that are rapidly proliferating (such as fast-growing cancer tumours). By inhibiting this step, cells die from a thymine less death because they have no thymine to create more DNA. This is often used in combination with Methotrexate, a potent inhibitor of dihydrofolate reductase enzyme.​

• Exemestane, a drug used in the treatment of breast cancer, is an inhibitor of the aromatase enzyme.​

Tumeric

Competitive inhibitors

• Compete with substrate for access to active site ​

• Often have similar structure to substrate​

• When bound to enzyme prevents binding of substrate ​

• Can be overcome by increasing [S] until it out-competes inhibitor

Diagram of CI

MM plot of CI on kinetics

Total final Vmax remains unchanged but higher S required for Vmax to be reached

Examples of CI

Other types of CI 

• Most useful therapeutic agents​

• Most drugs mimic transition states:​

• Bind enzymes 10 to the 3 – 10 to the 6 times tighter than natural substrates ​

• e.g. Tamiflu, Acarbose – Type II diabetes:

• ​

• Non-hydrolysable mimic of carbohydrates​

• Binds alpha-glucosidases – e.g. alpha-amylase​

• Prevents digestion of natural carbohydrates​

• Cheap, but not very effective on its own

• ​

• Statins – target hepatocytes and inhibit HMG-CoA reductase:​

• enzyme converts HMG-CoA into mevalonic acid, a cholesterol precursor. ​

• Statins bind 1000 x tighter than HMG-CoA​

NC inhibition

UC inhibition

Note

Learn the diagrams/graphs of three types of inhibition!

Learn Km and Vmax changes

What reaction conditions influence enzyme activity?

• Enzymes are sensitive to changes in their physical and chemical environments​

• Temperature​

• pH​

• Salts​

• Other chemicals​

• Molecular crowding​

pH on enzyme activity

Is optimum pH always neutral?

NO

Papain - enzyme activity flat between 4 + 8, denatures elsewhere

Choline esterase and pepsin - in diff acidic/basic environments so adapted to be at optimum in their environment

Temps effect on enzyme activity

Can also have optimums at different temperatures

Molecular crowding

Due to cell being crowded, enzymes and substrates may not be able to meet and have reactions, so this can be altered to increase activity