1. QUIZ 1.What is enzyme? 2.What is the function of enzyme? 3.What are the special characteristics of enzyme? 4.What kind of binding energy involve for the formation of ES complexes (enzyme-substrate complex)? 5.Define these terms: Active site Activation energy Cofactors Apoenzyme Holoenzyme Coenzyme Substrate

2. Lecture 2 – The Kinetics of Enzyme Catalyzed Reaction Dr. Saleha Shamsudin

3. Classification of Enzyme Enzymes fall into 6 classes based on function 1.Oxidoreductases: which are involved in oxidation, reduction, and electron or proton transfer reactions 2.Transferases : catalysing reactions in which groups are transferred 3.Hydrolases : which cleave various covalent bonds by hydrolysis 4.Lyases : catalyse reactions forming or breaking double bonds 5.Isomerases : catalyse isomerisation reactions 6.Ligases : join substituents together covalently.

5. SIMPLE ENZYME KINETICS Enzyme kinetics deals with the rate of enzyme reaction Kinetic studies of enzymatic reactions provide information about : (1)the basic mechanism of the enzyme reaction and (2) other parameters that characterize the properties of the enzyme. The rate equations developed from the kinetic studies can be applied in : (1)calculating reaction time, (2) yields, and (3) optimum economic condition, which are important in the design of an effective bioreactor. Enzyme kinetics deals with the rate of enzyme reaction Kinetic studies of enzymatic reactions provide information about : (1)the basic mechanism of the enzyme reaction and (2) other parameters that characterize the properties of the enzyme. The rate equations developed from the kinetic studies can be applied in : (1)calculating reaction time, (2) yields, and (3) optimum economic condition, which are important in the design of an effective bioreactor.

6. Assume that a substrate (S) is converted to a product (P) with the help of an enzyme (E) in a reactor as: If you measure the concentrations of substrate and product with respect to time, the product concentration will increase and reach a maximum value, whereas the substrate concentration will decrease as shown in Figure 2.1

7. The rate of reaction can be expressed in terms of either the change of the substrate Cs or the product concentrations C P as follows: Brown (1902) proposed that an enzyme forms a complex with its substrate. The complex then breaks down to the products and regenerates the free enzyme. The mechanism of one substrate-enzyme reaction can be expressed as:

8. One of the original theories to account for the formation of the enzyme- substrate complex is the "lock and key" theory. The main concept of this hypothesis is that there is a topographical, structural compatibility between an enzyme and a substrate which optimally favors the recognition of the substrate as shown in Figure 2.3. The reaction rate equation can be derived from the preceding mechanism based on the following assumptions: 1. The total enzyme concentration stays constant during the reaction, that is, C EO = C ES + C E 2. The amount of an enzyme is very small compared to the amount of substrate. Therefore, the formation of the enzyme substrate complex does not significantly deplete the substrate. 3. The product concentration is so low that product inhibition may be considered negligible.

9. Various study using x-ray and Raman spectroscopy have revealed the presence of enzyme-substrate complex. The simplest model describing this interaction is the lock- and-key model, in which the enzyme represents the lock and substrate represents the key. In multisubstrate, enzyme-catalyzed reactions, enzymes can hold substrates such that reactive regions of substrates are close to each other and to the enzyme’s active site, which is known as the proximity effects (nearest in distance). Also, enzymes can hold substrates at certain positions and angles to improve the reaction rate, which is known as the orientation effect.

10. Enzyme Kinetics Enzymes are protein catalysts that, like all catalysts, speed up the rate of a chemical reaction without being used up in the process. A mathematical model of the kinetics of single –substrate-enzyme-catalyzed reaction was first developed by V.C.R. Hendry (1902), and by L. Michaelis and M.L. Menten (1913). Kinetics of simple enzyme catalyzed reaction are often refered to Michaelis-Menten or saturation kinetics.

11. This model are based on data from batch reactors with constant liquid volume in which the the initial substrate,[S0], and enzyme [E0], concentration are known. An enzyme solution has a fixed number of active sites to which substrate can bind. At high substrate concentrations, all these sites may be occupied by substrates or the enzyme is saturated. Two major approaches used in developing a rate expression for enzyme catalyzed reactions are, (1) rapid-equilibrium approach and (2) quasi- steady-state approach.

13. Both quasi-steady state approximation and the assumption of rapid equibriuum share the same few steps in deriving a rate expression, Since the enzyme is not consumed, the conservation equation yields, Rate of product formation: Rate of variation of the ES complex:

15. Next topic: quasi-steady-state approach.