1. Enzymes Lab # 7

2. Enzymes: Definition Enzymes are highly specific biologic catalysts that greatly speed up the rate of a chemical reaction occurring in living cells Unit of enzymes activity International unit (IU) The amount of an enzyme that convert one micro-mole of a substrate per minute in an assay system

3. Measurement of enzymes activity Quantitations of enz. involve measurement of its catalytic activity & relate this activity to enz. conc. Look for - ↑ in product conc. - ↓ in substrate conc. - ↓ in coenzyme conc. - ↑ in altered coenzyme conc. Pyruvate + NADH +H + LDH Lactate + NAD +

4. Techniques for determination of enzyme activity I. Two-point assay (fixed time reaction) 1- incubate sample with substrate for a fixed time 2- stop the reaction 3- measure the amt. of product or substrate II. Kinetic or rate reaction assay (continuous monitoring) Multiple measurements of absorbance are made during the reaction

5. Kinetic or rate reaction assay: The use of coenzymes Example: NAD and NADH Determination of LDH activity: Pyruvate + NADH + H+ → lactate + NAD+

6. The extension of NAD and NADH Wave Length (nm) Absorbance NAD NADH 256 340 1.5 1

7. Enzymes classification Oxidoreductases -aerobic oxidases ex. tyrosinase -aerobic dehydrogenases ex. glucose oxidase -anaerobic dehydrogenases ex. LDH Transferases: ex. aminotransferases Hydrolases: ex.lipase Lyases: ex. decarboxylases Isomerases: ex. racemases Ligases or synthetases

8. Factors that influence enzymatic reaction Substrate concentration Enzyme concentration Product concentration PH Temperature Activators & Co-enzymes Inhibitors Specificity of enzymes