2. Enzyme activity is measured by the amount of product produced or the amount of substrate consumed. The rate of the enzymatic reaction is measured by the initial velocity v i. (velocity at the beginning of the reaction). The initial velocity is equal to the μmoles of product produced per min. Thus the higher the enzyme activity the higher the rate of the enzymatic reaction (v i ). It is a very important parameter to measure when studying enzymatic reaction. It is determined from the slope of the progress curve at the beginning of the reaction

3. Initial velocity

4. Velocity decreases with time because : (a) Substrate molecules may be used up. (b) Products may inhibit reaction (c) Change of pH (d) Cofactor or coenzyme may be used up. (e) Enzyme may lose activity

5. Factors that affect the rate of enzymatic reactions; 1- Substrate concentration. 2-Enzyme concentration. 3-pH. 4-Temperature.

6. Enzymes 1-Substrate concentration: The relationship between substrate concentration and rate of an enzymatic reaction is represented by a hyperbolic curve. a) At low substrate concentrations the rate of the enzymatic reaction is directly proportional to the substrate concentration (since many active sites are available for the substrate to bind to ). b) At medium substrate concentrations the relationship deviates from linearity, since less active sites are available. c)At high substrate concentrations the rate of the enzymatic reaction reaches maximum values and remains constant even with increased substrate concentrations,since all the active sites of the enzyme are saturated by the substrate (saturation effect).

7. Saturation of the enzyme binding sites

8. Effect of substrate concentration The rate of the enzymatic reaction at the saturation effect (the plateau part of the curve) reaches a maximum value and is called the Vmax. Km, the Michealis Constant is defined as the substrate concentration at half the Vmax. Km value reflects the affinity of the enzyme towards the subsrate. Km is inversely related to the affinity. The Km value is constant for an enzyme, substrate couple.

9. 2. Effect of temperature: Like most chemical reactions, the rate of an enzyme-catalyzed reaction increases as the temperature is raised. Higher temperature generally causes more collisions among the molecules and therefore increases the rate of a reaction. More collisions increase the likelihood that substrate will collide with the active site of the enzyme, thus increasing the rate of an enzyme-catalyzed reaction(for molecules to react, they have to collide with an energy equal to or greater than the activation energy for the reaction). A ten degree Centigrade rise in temperature will increase the activity of most enzymes by 50 to 100%. In the case of enzymatic reactions, this is complicated by the fact that many enzymes are adversely affected by high temperatures,the reaction rate increases with temperature to a maximum level (optimum temperature), then abruptly declines with further increase of temperature, due to the denaturation of the enzyme protein leading to it losing its catalytic activity.

10. Effect of temperature

11. 3. Effect of enzyme concentration: Increasing the enzyme concentration will increase the rate of the enzymatic reaction, this is so because when more enzyme molecules are present, more substrate molecules can be acted upon at the same time producing more products (providing that the substrate concentration is limitless). 4. Effect of PH: The point where the enzyme is most active - is known as the optimum pH.Extremely high or low pH values generally result in complete loss of activity for most enzymes caused by the enzyme denaturation. A change in pH can alter the ionization of the R-groups of amino acids. When the charges on the amino acids change, hydrogen and ionic bonds within the protein molecule change, this will lead to a consequent change in the enzyme shape (conformation), affecting its activity.

12. Effect of pH

13. Enzyme Inhibition Inhibitors are chemicals that reduce the rate of enzymatic reactions. Some are considered toxins, some are used as drugs. There are two classes of inhibitors ; 1-Irreversible inhibitors. 2-Reversible inhibitors. Irreversible Inhibitors; -They bind covalently (tightly) to the enzyme. -They permanently inactivate the enzyme thus they are called suicidal inhibitors.. -Many of them are toxins. Example 1: diisopropylfluorophosphate (DFP) binds covalently to serine in serine proteases &acetylcholinesterase. Example: nerve gas

14. Reversible inhibitors  Reversible inhibitors bind reversibly to the enzyme.  They bind non-covalently to the enzyme.  Types of reversible inhibitors include; a) Competitive inhibitors. b) Non-Competitive inhibitors. c) Uncompetitive inhibitors. Competitive inhibitors; -- Resembles the substrate’s structure closely. -They bind at the enzymes active site, thus competing with the substrate for the enzymes active site. -The inhibition can be overcome by increasing substrate concentration. -The inhibitor binds to the free enzyme forming an inactive EI complex.

15. Competitive inhibition

16. Non-competitive inhibitors They bind reversibly to the enzyme. They bind non-covalently to the enzyme. They bind to a site on the enzyme other than the active site. It can bind to the free enzyme and the ES complex. This action results in a conformational change in the protein that affects a catalytic step and hence decreases the enzyme activity. E + I EI ES + I ESI Both the EI and the ESI complex are inactive And cannot produce the product.

17. Uncompetitive Inhibition The inhibitor binds reversibly to the enzyme. The inhibitor binds non-covalently to the enzyme. It binds to a site other than the active site. It binds to the ES complex only which indicates that the inhibitors site is exposed and available only when the enzyme binds the substrate. ES + I ESI, which is an inactive complex that does not produce products.

18. Allosteric enzymes They are regulatory enzymes. They have an active site and a regulatory (modulatory) site. They usually have a quatarnary structure. The active site binds the substrate, while the modulatory site binds to certain modulators, -ve modulators will decrease the enzyme activity, while +ve modulators will increase the enzyme activity. They usually catalyze irreversible reactions. They are important in regulating metabolic pathways.