Rates of Enzyme Reactions

Most biochemical reactions in living systems are catalyzed by enzymes - that is, biocatalysts - which includes proteins and, in some cases, cofactors and coenzymes such as vitamins, nucleotides, and metal cations. Enzyme-catalyzed reactions generally proceed via intermediates, for example. 

The reactants in enzyme reactions are known as substrates. Enzyme reactions may involve uni-, bi-, or trimolecule reactants and products. An analysis of the reaction kinetics of such complicated enzyme reactions, however, is beyond the scope of this chapter, and the reader is referred elsewhere [1] or to other reference books. Here, we shall treat only the simplest enzyme-catalyzed reaction - that is, an irreversible, unimolecular reaction. 

We consider that the reaction proceeds in two steps, namely, 

Michaelis-Menten Approach In enzyme reactions, the total molar concentration of the free and combined enzyme, Cpg (kmol m ) should be constant that is. 

Eor the first reaction at equilibrium the rate of the forward reaction should be equal to that of the reverse reaction, as stated in Section 3.2.I.I. 

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The rate of enzyme reaction is often described by the Michaelis-Menten equa-... 

Rates of enzyme reactions are often affected by the presence of various chemicals and ions. Enzyme inhibitors combine, either reversibly or irreversibly, with enzymes and cause a decrease in enzyme activity. Effectors control enzyme reactions by combining with the regulatory site(s) of enzymes. There are several mechanisms of reversible inhibition and for the control of enzyme reactions. 

Assuming that the local rate of enzyme reaction follows Michaelis-Menten kinetics, or that the microbe film follows Monod kinetics regardless of immobilisation, then equation 5.86 becomes ... 

Enzyme kinetics deals with the rate of enzyme reaction and how it is affected by various chemical and physical conditions. Kinetic studies of enzymatic reactions provide information about the basic mechanism of the enzyme reaction and other parameters that characterize the properties of the enzyme. The rate equations developed from the kinetic studies can be applied in calculating reaction time, yields, and optimum economic condition, which are important in the design of an effective bioreactor. 

If enzymes are immobilized by copolymerization or microencapsulation, the intraparticle mass-transfer resistance can affect the rate of enzyme reaction. In order to derive an equation that shows how the mass-transfer resistance affects the effectiveness of an immobilized enzyme, let s make a series of assumptions as follows ... 

When Rc = 0, assume the value of xSq atf = 0. This is the case when the rate of enzyme reaction is slow compared to that of mass transfer which is represented by the low value of phi. As a result, the substrate reaches the center of sphere. 

Since microbial activity and growth are manifestations of enzymatic action, and since the rates of enzyme reactions increase... 

Enzyme activity may be inhibited by substances that inactivate the enzyme or occupy the active site of the enzyme before the substrate has a chance. As a result, the rate of transformation of the substrate to product is slowed. In competitive inhibition, similar substrates (or analogs) can bind to the same active site on the enzyme. Therefore, they compete with each other for the same active sites. This inhibition process is reversible and can be prevented or slowed by increasing the substrate concentration or by diluting the inhibitor in the solution. In this case, the enzyme already bound to the substrate is not inhibited. The effect of the competitive inhibitor (I) on the rate of enzyme reaction in Equation (5.129), Equation (5.130), Equation (5.131), and Equation (5.132) yields ... 

EMIT Using Hapten-Enzyme Conjugate. In this assay, a fixed amount of hapten-enzyme conjugate is incubated with a fixed amount of antibody to the hapten together with a variable amount of free hapten. The antibody will cause a change in enzymic activity upon reaction with one or more haptens on the enzyme. In this assay, equilibrium does not necessary have to be attained before enzyme measurement, no separation of free from antibody-bound hapten is required, and the rate of enzyme reaction is measured, rather than an end point. 

Activity assays of enzymes bound to solid phases in EIA systems have previously been limited to fixed-time spectrophotometric methods following incubation of substrate and solid phase for extended periods of time. Kinetic assays of enzyme activity have not been used to date because of the difficulty in directly monitoring initial rates of enzyme reactions in a turbid solid phase suspension. With urease as the label, an ammonia gas sensing electrode can be used to directly quantitate the amount of urease-labeled antigen or hapten bound to a double-antibody solid phase by continuously measuring the initial rate of ammonia produced from urea as a substrate. 

As can be seen from reaction (1), oxygen is an integral part of the generation of NO fi om L-arginine. Experiments have been carried out into the effect of oxygen concentration on the rate of enzyme reaction. 

As in heterogeneously catalyzed processes, the rate of enzyme reactions usually follow saturation kinetics with respect to the concentration of S as shown in Fig. 7.1. This curve is redrawn in Fig. 7.2 to show the relationship to and K]vi which is the substrate concentration when v = 1/2 V ax- At low values of [S]... 

The rate of enzyme reactions rises with temperature up to a certain optimum. Above that, the effect of thermal inactivation dominates over that of the increase of the collision frequency. 

The temperature at which food is held can have a great influence on the kind, rate, and amount of microbiological changes that would take place. The growth and metabolic reactions of microorganisms depend on enzymes the rates of enzyme reactions are directly affected by temperature. Destruction of microorganisms at high temperature is supposed to be caused by denaturation of proteins or nucleic acid and loss of cytoplasmic membrane function (Perry and Staley, 1997). 

In most cases the rates of enzyme reactions pass through a maximum as the pH is varied, as shown in Figure 10.7. The pH corresponding to the maximum rate is known as the optimum pH. The optimum pH is sometimes regarded as a characteristic property of an enzyme however, it varies somewhat with the nature of the substrate and with the substrate concentration. 

Enhancement of the rates of enzyme reactions by microwave irradiation has a so been reported by Chen et al. [89] who studied enzyme activity in the regioselective acylation of sugar derivatives in nonaqueous solvents. 

It has been observed that the rate of enzyme reaction rises with temperature up to a certain maximum above which, thermal inactivation of the enzyme takes place. The inactivation of enzymes by heat is due to the denaturation of the enzyme protein. The effect of the instability of the enzyme, free and in the immobilised state, can be studied by exposing the enzyme to thermal treatment for a defined period prior to measuring its activity at a temperature at which it is stable. Chaubey and co-workers obtained 40 °C as the critical temperature of PPy-polyvinyl sulfonate films immobilised with crosslinked lactate dehydrogenase [123]. The activation energies below and above the critical temperature were found to be 93.3 and 22.4 kj/mole, respectively. 

Studies on the effects of adenine nucleotides on the reaction for barley leaf PQK isoenzymes have revealed that these isoenzymes are not simply regulated by energy charge, but the nucleotides behave more as allosteric regulators. Fig.4 shows the effects of ADP and AMP on the reaction rates observed for the barley isoenzymes as compared with yeast, it can be seen that in all cases, the nucleotide analogues inhibit the rate of enzyme reaction to a greater extent in the plant isoenzymes as compared with the yeast enzyme and that the chloroplast isoenzyme showed most inhibition. Extensive studies on the effects of adenine nucleotides on the yeast enzyme (3,11,15) revealed that there appeared to be at least two different sites for the binding of adenine nucleotides. 

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