1. CHAPTER 1: ENZYME KINETICS AND APPLICATIONS (Part Ib : Kinetics of Enzyme Catalyzed Reactions) ERT 317 Biochemical Engineering Sem 1, 2015/2016

2. Models for More Complex Enzyme Kinetics

3. Allosteric Enzymes Allostery @ cooperative binding The binding of one substrate to enzyme facilitates binding of other substrate molecules Rate expression: Where, n = cooperativity coefficient : n>1 indicates positive cooperativity (activator; n<1=inhibitor) The cooperativity coefficient can be determined by rearranging above equation

4. Graphical Determination of the Cooperativity Coefficient, n

5. Comparison of Michaelis-Menten and Allosteric Enzyme kinetics The binding of one substrate to enzyme facilitates binding of other substrate molecules

6. Inhibited Enzyme Kinetics ENZYME INHIBITORS : Certain compound that bind to enzyme and reduce enzyme activity May be IRREVERSIBLE or REVERSIBLE IRREVERSIBLE ( like heavy metal)form a stable complex with enzyme and reduce enzyme activity Such enzyme inhibition may be REVERSED only by using chelating agents such as EDTA and citrate. It is easily DISSOCIATED from the enzyme after binding.

7. 1. COMPETITIVE 2. NONCOMPETITIVE 3. UNCOMPETITIVE 3 Major Classes REVERSIBLE Enzyme Inhibition

8. K’ m,app The effect of such inhibitors can be overcome by increasing the substrate concentration. Equation for the rate of enzymatic conversion 1. Competitive Inhibition Substrate analogs and compete with substrate for the active site of the enzyme

9. Noncompetitive inhibitors bind on site other than the active site, reduce enzyme affinity to the substrate Can be described by : Rate equation: Overcome by adding reagents to block binding of inhibitor 2. Noncompetitive Inhibition

10.  Inhibitors binds to the ES complex only and have no affinity for the enzyme itself  Rate of reaction,  The net effect is reduction in both V m and K’ m values. 3. Uncompetitive Inhibition

11. Substrate inhibition High substrate concentrations may cause inhibition in some enzymatic reaction

13. The Effects of Enzyme Inhibitors Substrate analogs and compete with substrate for the active site of the enzyme Noncompetitive inhibitors bind on site other than the active site, reduce enzyme affinity to the substrate

14. Effect of pH and temperature

15. Effect of pH on Enzyme Kinetics -Enzyme are active only over small range of pH due to: the active site functional group charges (ionic form) the three dimensional shape of enzyme are pH- dependent -these ionic group on active sites must be in a suitable form (acid or base) to function. -Variation in pH of medium result in changes of: Ionic form of the active site Activity of enzyme, hence the reaction rate Affect the maximum reaction rate, K m and enzyme stability

16. -Scheme to describe pH dependence of the enzymatic reaction rate for ionizing enzymes. -Optimum pH is usually determined experimentally Variation of enzyme activity with pH for 2 different enzymes (A) and (B) - Ionic groups at active site

17. Effect of Temperature on Enzyme Kinetics

18. Variation of reaction rate with temperature ascending descending The rate varies according to Arrhenius equation Thermal denaturation occurred

19.  k d is the denaturation constant,  Enzyme denaturation is much faster than enzyme activation.  Variation in T affect both V m and K m The rate,

20. Thank You MID TERM EXAM 1 week 6 FRIDAY, 16 Oct 2015 (DKD1) 9-11 am