1. Enzyme Functions and Regulations 1

2. Exer Biochem c2-enzyme 2 Enzymes as catalysts 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 3 酵素的作用與能量釋放

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

5. Exer Biochem c2-enzyme 5 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 6

7. 7 Isozymes, isoenzymes 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 8 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 9 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 10 pH and temperature affect enzyme velocity

11. Exer Biochem c2-enzyme 11 Turnover number (k cat ) Turnover number (k cat ), 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 10 6 -10 7 Usually use kcat/Km: how fast the enzyme can work under physiological conditions Far less diversified

12. Exer Biochem c2-enzyme 12 Enzyme inhibition 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 13

14. Exer Biochem c2-enzyme 14 Enzyme cofactors 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 15

16. Exer Biochem c2-enzyme 16

17. Exer Biochem c2-enzyme 17 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 18 Active transport: creatine transporter in muscle cells

19. Exer Biochem c2-enzyme 19 Oxidation and reductions: redox reactions 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 20

21. Exer Biochem c2-enzyme 21

22. Exer Biochem c2-enzyme 22 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 23

24. Exer Biochem c2-enzyme 24 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 25

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

27. Exer Biochem c2-enzyme 27 Phosphorylation/dephosphorylation

28. Exer Biochem c2-enzyme 28 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 29 irreversible

30. Exer Biochem c2-enzyme 30

31. Exer Biochem c2-enzyme 31 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 32 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