Enzyme Functions and Regulations

1. Enzyme Functions and Regulations

2. Exer Biochem c2-enzyme Enzymes as catalysts 催化劑 • Enzymes are proteins that catalyze different chemical reactions that constitute our metabolism • Speed up chemical reactions by lowering their energy barrier (energy of activation), so that the reaction can take place at low temperature (37C) • May increase reaction speed by millions of times • Active site: AA residues that bind substrates and perform catalysis • Binding site, catalytic site • usually clefts in 3-D structure with specific AA residues to interact with substrate 受質

3. Exer Biochem c2-enzyme 酵素的作用與能量釋放

4. Exer Biochem c2-enzyme 酵素作用模型 lock and key

5. Exer Biochem c2-enzyme Rates of enzymatic reactions:Substrate concentration • Initial velocity: enzyme reaction rate has to be measured quickly before accumulation of products • Plot Initial velocity vs [S] • Usually hyperbolic (but not all), Michaelis-Menten kinetics • Vmax: maximum velocity • Enzyme active sites are totally saturated • Michaelis constant (Km): [S] at ½ Vmax • Affinity of enzyme for its substrate: ↑Km, ↓affinity • [S] usually ≦ Km in cells, quicker response to changes in [S] • calculated more easily by Lineweaver-Burk plot

6. Exer Biochem c2-enzyme

7. Exer Biochem c2-enzyme Isozymes, isoenzymes 同工酶 • Enzymes catalyze same reaction in different tissues, but have different kinetic parameters • Hexokinase I, II, III, IV • Low-Km isozymes function when [glucose] is low, especially in brain • High-Km isozymes in liver, high [glucose]

8. Exer Biochem c2-enzyme Rates of enzymatic reactions:enzyme concentration • ↑[enzyme], ↑Vmax in proportion • No effect on Km • ↑certain enzyme concentrations after exercise training • adaptation

9. Exer Biochem c2-enzyme Environmental effects on enzyme function • Optimal pH • Changes in active site • or changes other sites that affect active sites • Optimal temperature • Temperature too high cause protein denaturation • Loss of enzyme activity • Warming up muscles prior to exercise increase enzyme activities

10. Exer Biochem c2-enzyme pH and temperature affect enzyme velocity

11. Exer Biochem c2-enzyme Turnover number (kcat) • Turnover number (kcat), catalytic constant • Maximum number of substrate converted to product per enzyme active site per unit of time (usually per sec) • How fast an enzyme can convert substrate to product, Maximum catalytic activity for enzyme • Very diversified among different enzymes, can reach 106-107 • Usually use kcat/Km: how fast the enzyme can work under physiological conditions • Far less diversified

12. Exer Biochem c2-enzyme Enzyme inhibition 抑制 • Competitive inhibitors • Resemble normal substrate • Bind to active site, but can not be changed into product • Noncompetitive inhibitors • Does not resemble normal substrate • Does not bind to active site • When bind to enzyme, it interferes with enzyme function

13. Exer Biochem c2-enzyme

14. Exer Biochem c2-enzyme Enzyme cofactors 輔酶 • Enzymes may need other reactive groups (not AA) for their functions • Cofactors • May be metal ions, Mg, Zn, Mn… • May be organic molecules: coenzymes (e.g. NAD, FAD) • Apoenzyme (inactive) + cofactor = holoenzyme (active) • Prosthetic group: a cofactor tightly bound to the enzyme at all times (e.g. heme in hemoglobin) • Deficiency diseases associated with inadequate intake of specific vitamins may due to insufficient catalytic power of enzymes

15. Exer Biochem c2-enzyme

16. Exer Biochem c2-enzyme

17. Exer Biochem c2-enzyme Protein transporters • Transmembrane proteins, integral membrane proteins • Technically not enzymes, but function consistently with the kinetics of enzymes (Vmax, Km) • Recognize specific transport substances, move them in a particular direction • Specific transport proteins: translocase, porter, carrier, transporter, channel • Facilitated diffusion: from higher to lower concentration • Active transport: from lower to higher concentration • Require energy: ATP or other chemical gradients • Creatine transporter in muscle cell

18. Exer Biochem c2-enzyme Active transport: 主動運輸creatine transporter in muscle cells

19. Exer Biochem c2-enzyme Oxidation and reductions: redox reactions 氧化還原反應 • Oxidation: something lose electrons • Reduction: something gain electrons • These 2 reactions are always connected • Dehydrogenation: hydrogen leaves as electrons • Oxidation reactions • Coenzymes that accept hydrogen (electron) • NAD+  NADH; FAD  FADH2

20. Exer Biochem c2-enzyme

21. Exer Biochem c2-enzyme

22. Exer Biochem c2-enzyme Regulation of enzyme activity • Control biological functions through control of enzyme activities • Synthesis/degradation of enzyme is time- and energy-consuming • Allosteric enzymes (allo: other), usually have subunits • Enzymes with sites other than active site that effectors can bind and modify enzyme activity: allosteric sites • Usually in metabolic pathways where they can control the rate of flux of the entire pathway (rate-limiting enzymes, e.g. phosphofructokinase) • Ligand: molecule that bind to large molecules • Ligands can bind to allosteric sites and increase or decrease enzyme activity

23. Exer Biochem c2-enzyme

24. Exer Biochem c2-enzyme Regulation of enzyme activity • Ratio of positive/negative effectors (activator/inhibitor) • Feedback inhibition • Enzyme activity inhibited by its product • Allosteric regulation is ‘fine-tuning’ type of enzyme activity modulation

25. Exer Biochem c2-enzyme

26. Exer Biochem c2-enzyme Regulation of enzyme activity: Phosphorylation, dephosphorylation • Covalent modification of enzymes • Rapidly turn on or off enzyme activity • Phosphorylation磷酸化, dephosphorylation 去磷酸化 • Add/remove a phosphate group in specific AA • Catalyzed by protein kinase, phosphoprotein phosphatase • Critical in controlling and integrating metabolism, signal transduction pathways • Controlled by hormones, cytokines, other factors

27. Exer Biochem c2-enzyme Phosphorylation/dephosphorylation

28. Exer Biochem c2-enzyme Regulation of enzyme activity: Thiol oxidation and reduction • Thiol oxidation and reduction: redox control of enzyme functions • Usually on cysteine thiols (-SH) • reactive oxygen species (superoxide), reactive nitrogen species (nitric oxide) • When proteins are oxidized: thiols may form sulfenic (P-SOH) , sulfinic (P-SOzH), or sulfonic (P-S03H) acids; intra- or interprotein disulfides (P-S-S-P); nitrosothiols (P-SNO), glutathione (P-S-SG) • can be reversed by specific protein-reducing enzymes called glutaredoxins, thioredoxins, peroxiredoxins

29. Exer Biochem c2-enzyme irreversible

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31. Exer Biochem c2-enzyme Measurement of phosphorylated proteins • Western blot • Serine, tyrosine, threonine • Serine kinase, tyrosine kinase • Antibody to phospho-serine, phospho-tyrosine • Or specific Phosphorylated protein • Phosphorylated/total • Total GS, phophorylated GS (203-Ser phosphorylated GS, 872-Thr-phosphorylated GS) • Stripping buffer

32. Exer Biochem c2-enzyme Measurement of enzyme activity • Usually measure maximal activity • [S] high enough to generate true Vmax • Standardized pH, temperature • Simple method to measure [S] or [P], e.g. color • Use NADH disappearance/appearance • Absorption at 340 nm • One international • 1 international unit (IU) of enzyme activity:the amount of enzyme that converts one micromole of substrate to product in one minute • Usually IU/mg tissue, IU/ml