Enzyme kinetics by kk sahu sir

History
Introduction
General Principles
Enzyme Assays
Single Substrate Reactions
Substrate Complex
The Velocity equation can be derived from following method
Lineweaver-Burk Plot
Derivation
Enzyme Kinetics as an Approach to Understanding Mechanism
References

1. Enzyme Kinetics 1 By KAUSHAL KUMAR SAHU Assistant Professor (Ad Hoc) Department of Biotechnology Govt. Digvijay Autonomous P. G. College Raj-Nandgaon ( C. G. )

2. Contents History Introduction General Principles Enzyme Assays Single Substrate Reactions Substrate Complex The Velocity equation can be derived from following method Lineweaver-Burk Plot Derivation Enzyme Kinetics as an Approach to Understanding Mechanism References 2

3. History In 1902  Victor Henri  proposed a quantitative theory of enzyme kinetics, but at the time the experimental significance of the hydrogen ion concentration was not yet recognized. After  Peter Lauritz Sørensen  had defined the logarithmic pH-scale and introduced the concept of buffering in 1909 the German chemist  Leonor Michaelis  and his Canadian postdoc  Maud Leonora Menten repeated Henri's experiments and confirmed his equation, which is now generally referred to as  Michaelis-Menten kinetics .  Their work was further developed by  G. E. Briggs  and  J. B. S. Haldane , who derived kinetic equations that are still widely considered today a starting point in modeling enzymatic activity. 3

4. Introduction Enzyme kinetics  is the study of the chemical reactions that are catalysed by enzymes. In enzyme kinetics, the reaction rate is measured and the effects of varying the conditions of the reaction are investigated. Enzymes are usually protein molecules that manipulate other molecules—the enzymes' substrates. These target molecules bind to an enzyme's active site and are transformed into products through a series of steps known as the enzymatic mechanism. E + S ⇄ ES ⇄ ES* ⇄ EP ⇄ E + P 4

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6. General Principles The reaction catalysed by an enzyme uses exactly the same reactants and produces exactly the same products as the uncatalysed reaction. Like other catalysts, enzymes do not alter the position of equilibrium between substrates and products. However, unlike uncatalysed chemical reactions, enzyme-catalysed reactions display saturation kinetics. For a given enzyme concentration and for relatively low substrate concentrations, the reaction rate increases linearly with substrate concentration; the enzyme molecules are largely free to catalyse the reaction, and increasing substrate concentration means an increasing rate at which the enzyme and substrate molecules encounter one another. However, at relatively high substrate concentrations, the reaction rate asymptotically approaches the theoretical maximum; the enzyme active sites are almost all occupied and the reaction rate is determined by the intrinsic turnover rate of the enzyme. 6

7. Fig. As larger amounts of substrate are added to a reaction, the available enzyme binding sites become filled to the limit. Beyond this limit the enzyme is saturated with substrate and the reaction rate increase. 7

8. Enzyme Assays Enzyme assays are laboratory procedures that measure the rate of enzyme reactions. Since enzymes are not consumed by the reactions they catalyse, enzyme assays usually follow changes in the concentration of either substrates or products to measure the rate of reaction. There are many methods of measurement.  Spectrophotometric assays observe change in the absorbance of light between products and reactants; radiometric assays involve the incorporation or release of radioactivity to measure the amount of product made over time. Spectrophotometric assays are most convenient since they allow the rate of the reaction to be measured continuously. Although radiometric assays require the removal and counting of samples (i.e., they are discontinuous assays) they are usually extremely sensitive and can measure very low levels of enzyme activity. 8

9. Fig. Progress curve for an enzyme reaction. The slope in the initial rate period is the  initial rate of reaction   v . The Michaelis–Menten equation describes how this slope varies with the concentration of substrate. 9

10. Single-Substrate Reactions Michaelis–Menten kinetics As enzyme-catalysed reactions are saturable, their rate of catalysis does not show a linear response to increasing substrate. If the initial rate of the reaction is measured over a range of substrate concentrations (denoted as [S]), the initial reaction rate increases as [S] increases, as shown on the right. However, as [S] gets higher, the enzyme becomes saturated with substrate and the initial rate reaches  V max , the enzyme's maximum rate. 10

11. Fig. A chemical reaction mechanism with or without enzyme catalysis. The enzyme (E) binds substrate (S) to produce product (P). 11

12. There is an initial bimolecular reaction between the enzyme E and substrate S to form the enzyme–substrate complex ES. The rate of enzymatic reaction increases with the increase of the substrate concentration up to a certain level called V max ; at V max , increase in substrate concentration does not cause any increase in reaction rate as there no more enzyme (E) available for reacting with substrate (S). Here, the rate of reaction becomes dependent on the ES complex and the reaction becomes a  unimolecular reaction with an order of zero. 12

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23. Enzyme Kinetics as an Approach to Understanding Mechanism Biochemists commonly use several approaches to study the mechanism of action of purified enzymes. The three dimensional structure of the protein provides important information, which is enhanced by classical protein chemistry and modern methods of site-directed mutagenesis. These technologies permit enzymologists to examine the role of individual amino acids in enzyme structure and action. However, the oldest approach to understanding enzyme mechanisms, and the one that remains most important, is to determine the rate of a reaction and how it changes in response to changes in experimental parameters, a discipline known as enzyme kinetics. 23

24. References Biochemistry – Lehninger ( Nelson and Cox) Sixth Edition Internet – www.google.com 24