1. Mr. Halavath Ramesh 16-MCH-001 Department of Chemistry Loyola College-Chennai University of Madras

2. Introduction: Enzymes are very efficient catalysts for biochemical reactions. They speed up reactions by providing an alternative reaction path way of lower activation energy. Enzymes are widely used commercially for example in the detergent food and brewing industries. Protease enzymes are used in biological washing powders to speed up the break down of proteins in stains like blood and egg. The use of enzymes in the diagnosis is one of the important benefits derived from the intensive research in biochemistry since the 1940’s.Enzymes have provided the basis for the field of clinical chemistry. The living cell is the site of tremendous activity called Metabolism. Chemical Nature of Enzymes: All known enzymes are proteins. They are high molecular weight compounds made up principally of chains of amino acids linked together by peptide bonds. Enzymes can be denatured and precipitated with salts, solvents and other reagents. They have molecular weights ranging from 10,000 to 2,000,000. Most chemical catalysts catalyze a wide range of reactions. They are not usually very selective. In contrast enzymes are usually highly selective, catalyzing specific reactions only. This specificity is due to the shapes of the enzyme molecules. For two molecules to react they must collide with one another. They must collide in the right direction (orientation) and with sufficient energy. Sufficient energy means that between them they have enough energy to overcome the energy barrier to reaction. This is called the activation energy.

3. Many enzymes require the presence of other compounds - cofactors - before their catalytic activity can be exerted. This entire active complex is referred to as the holoenzyme; i.e., apoenzyme (protein portion) plus the cofactor (coenzyme, prosthetic group or metal-ion- activator) is called the holoenzyme. Enzymes have an active site. This is part of the molecule that has just the right shape and functional groups to bind to one of the reacting molecules. The reacting molecule that binds to the enzyme is called the substrate. An enzyme-catalysed reaction takes a different 'route'. The enzyme and substrate form a reaction intermediate. Its formation has a lower activation energy than the reaction between reactants without a catalyst.

5. Factors affecting Catalytic activity of enzymes: Enzyme activity can be affected by a variety of factors, such as temperature, pH, and concentration. Enzymes work best within specific temperature and pH ranges, and sub-optimal conditions can cause an enzyme to lose its ability to bind to a substrate. Temperature: Raising temperature generally speeds up a reaction, and lowering temperature slows down a reaction. However, extreme high temperatures can cause an enzyme to lose its shape (denature) and stop working. As the temperature rises, reacting molecules have more and more kinetic energy. This increases the chances of a successful collision and so the rate increases. There is a certain temperature at which an enzyme's catalytic activity is at its greatest (see graph). This optimal temperature is usually around human body temperature (37.5 o C) for the enzymes in human cells. Above this temperature the enzyme structure begins to break down (denature) since at higher temperatures intra- and intermolecular bonds are broken as the enzyme molecules gain even more kinetic energy.

6. pH: Each enzyme has an optimum pH range. Changing the pH outside of this range will slow enzyme activity. Extreme pH values can cause enzymes to denature. Each enzyme works within quite a small pH range. There is a pH at which its activity is greatest (the optimal pH). This is because changes in pH can make and break intra- and intermolecular bonds, changing the shape of the enzyme and, therefore, its effectiveness. Enzyme Concentration: Increasing enzyme concentration will speed up the reaction, as long as there is substrate available to bind to. Once all of the substrate is bound, the reaction will no longer speed up, since there will be nothing for additional enzymes to bind to. Substrate concentration: Increasing substrate concentration also increases the rate of reaction to a certain point. Once all of the enzymes have bound, any substrate increase will have no effect on the rate of reaction, as the available enzymes will be saturated and working at their maximum rate.

7. Concentration of enzyme and substrate: The rate of an enzyme-catalysed reaction depends on the concentrations of enzyme and substrate. As the concentration of either is increased the rate of reaction increases (see graphs). For a given enzyme concentration, the rate of reaction increases with increasing substrate concentration up to a point, above which any further increase in substrate concentration produces no significant change in reaction rate. This is because the active sites of the enzyme molecules at any given moment are virtually saturated with substrate. The enzyme/substrate complex has to dissociate before the active sites are free to accommodate more substrate. Provided that the substrate concentration is high and that temperature and pH are kept constant, the rate of reaction is proportional to the enzyme concentration.

8. Specificity of Enzymes One of the properties of enzymes that makes them so important as diagnostic and research tools is the specificity they exhibit relative to the reactions they catalyze. A few enzymes exhibit absolute specificity; that is, they will catalyze only one particular reaction. Other enzymes will be specific for a particular type of chemical bond or functional group. In general, there are four distinct types of specificity: 1.Absolute specificity - the enzyme will catalyze only one reaction. 2. Group specificity - the enzyme will act only on molecules that have specific functional groups, such as amino, phosphate and methyl groups. 3. Linkage specificity - the enzyme will act on a particular type of chemical bond regardless of the rest of the molecular structure. 4. Stereo chemical specificity - the enzyme will act on a particular steric or optical isomer. Inhibition of enzyme activity Some substances reduce or even stop the catalytic activity of enzymes in biochemical reactions. They block or distort the active site. These chemicals are called inhibitors, because they inhibit reaction. Inhibitors that occupy the active site and prevent a substrate molecule from binding to the enzyme are said to be active site-directed (or competitive, as they 'compete' with the substrate for the active site). Inhibitors that attach to other parts of the enzyme molecule, perhaps distorting its shape, are said to be non-active site-directed (or non competitive).

10. Key Terms Catalyst: A substance that speeds up a chemical reaction without being changed. Enzyme: A biological catalyst(usually a protein) Substrate: The reactant molecules that an enzyme works on. Active site: The part of the enzyme where the substrate binds. Inhibitors Competitive Inhibitors Non- Competitive (Mixed) Inhibitors Uncompetitive Inhibitors

11. Basic Enzyme Reactions Enzymes are catalysts and increase the speed of a chemical reaction without themselves undergoing any permanent chemical change. They are neither used up in the reaction nor do they appear as reaction products. The basic enzymatic reaction can be represent as follows S + E ……………..> P + E Where E represent the enzyme catalyzing the reaction, S the substrate, the substance being changed and P the product of the reaction. Energy Levels: Chemists have known for almost a century that for most chemical reactions to proceed, some form of energy is needed. They have termed this quantity of energy, "the energy of activation." It is the magnitude of the activation energy which determines just how fast the reaction will proceed. It is believed that enzymes lower the activation energy for the reaction they are catalyzing. Figure 3 illustrates this concept. The enzyme is thought to reduce the "path" of the reaction. This shortened path would require less energy for each molecule of substrate converted to product. Given a total amount of available energy, more molecules of substrate would be converted when the enzyme is present (the shortened "path") than when it is absent. Hence, the reaction is said to go faster in a given period of time.

13. The Enzyme Substrate Complex: A theory to explain the catalytic action of enzymes was proposed by the Swedish chemist Savante Arrhenius in 1888. He proposed that the substrate and enzyme formed some intermediate substance which is known as the enzyme substrate complex. The reaction can be represented as: If this reaction is combined with the original reaction equation [1], the following results:

15. It is important to note that the enzymes do not change the equilibrium constant (Keq) or free energy change (∆G) of the reaction. Each enzyme present in a cell has its characteristic enzyme parameters. The plot of initial reaction velocity, V0 against the substrate concentration [S] has same general shape (rectangular hyperbolic shape) which is given by Michaelis-Menten equation:

16. where, V0 is the initial reaction rate, Vmax is the maximum rate, [S] is the molar substrate concentration, and Km is a constant called Michaelis constant. Vmax and Km are the characteristic properties of an enzyme. As is clear from equation 12.1, Km can be defined as the substrate concentration at which initial reaction rate, V0 equals 2. The response of enzymes to the concentrations of substrates and products plays important role in the reaction control. This behavior of enzymes to the substrate/product concentration is studied under enzyme kinetics and is used to determine the important enzyme parameters such as Km and Vmax. We have chosen to study the kinetics of the enzyme alkaline phosphatase. The enzyme catalyses the hydrolysis of a phosphoester bond, producing inorganic phosphate (Pi) and an alcohol. We have chosen p- nitrophenylphosphate as the substrate for the hydrolysis reaction. Para-nitrophenylphosphate is a colourless compound; the enzyme, alkaline phosphatase hydrolyses the phosphoester bond to produce the coloured product, pnitrophenol which can be detected colorimetrically (Figure 12.2)