Kinetics of enzyme-coenzyme reactions

Initial rate measurements, especially with alternative substrates and with a product or substrate analog as inhibitor, and measurements of the rate of isotope exchange at equilibrium, can give a great deal of information about mechanism, and in some cases allow estimates of individual velocity constants and dissociation constants. The results of such studies, which require little enzyme, are an essential basis for the proper interpretation, in relation to the overall catalytic reaction, of pre-steady-state studies and kinetic and thermodynamic studies of enzyme-coenzyme reactions in isolation. 

As in the Jones protocol the cubic section model of the substrate binding domain of HLADH were constructed using structures of alcohol products rather than ketone substrates. The alcohol products were originally chosen by the Jones group because the transition state the geometry for the reduction was considered to resemble that of the alcohol rather than that of the ketone. The relative rate of reduction of substrate vs cyclohexanone for each ketone was required to be known. Furthermore, configurations of alcohol products, enantiomeric excess values, yields and % conversion of substrate required for calculation of the priority number for each enantiomer of product should be measured under comparable conditions (i.e. pH, temperature, concentration of enzyme, coenzyme and substrate, etc.). According to Alderweireldt et al. (1988) HLADH models are valid only for reaction conditions used in the reactions from which the models are constructed. Furthermore, the model is oniy reliable if the reactions have been conducted under kinetic control. 

It should be noted that the relations in Eq. (8) are valid for this mechanism, however, because ad/< b is equal to the apparent dissociation constant, [E][A]/ [EA] - - [E A]1, which will also be measured in any direct equilibrium study of the enzyme-coenzyme reaction (Section III,A). Moreover, the functions of kinetic coefficients in Eqs. (5) and (6), as well as those in Eq. (8), should be independent of the nature of the second substrate B or Q, as in the case of the simple ordered mechanism, since they are defined by rate constants for the enzyme-coenzyme and isomerization reactions only (Section II,E). 

The main value of product inhibition studies of dehydrogenases has been to distinguish between ordered and random mechanisms and to provide additional kinetic estimates of the dissociation constants of enzyme-coenzyme compounds. On both counts the method has been especially useful for reactions that are essentially irreversible or for other reasons cannot be studied in both directions 122,138). It is also in such circumstances that product inhibition studies are most reliable because, as Alberty (7) emphasized when proposing the method, with readily reversible reactions it may be difficult to estimate true initial rates with small concentrations of substrates in the presence of a product. The reality of ternary complexes in an ordered mechanism of the Theorell-Chance type has also been demonstrated with several enzymes (134) by product inhibition studies. 

For the four mechanisms described in Section II,B the dissociation constants of the binary coenzyme complexes can be calculated from kinetic coefficients for the overall reaction by Eq. (8). Such estimates are compared in Tables IV-VI (158-162a) with direct estimates from studies of enzyme-coenzyme equilibria at similar pH values and temperatures. In comparing these values, it must be borne in mind that 0ab and < pq are estimated from initial rates with small concentrations of coenzyme... 

In the oxidation of secondary alcohols by DADH, the coenzyme is the leading substrate, the release of NADH from the enzyme-NADH complex is the rate-limiting step, and the maximum velocity vmax is independent of the chemical nature of the alcohol. In the case of primary alcohols, as vmax is much lower and depends on the nature of the alcohol, Theorell-Chance kinetics (Figure 9.9) are not observed and the rate-limiting step is the chemical interconversion from alcohol to aldehyde. With all this biochemical information it is possible to delineate a catalytic reaction mechanism that is in agreement with the crystal structures and the steps of alcohol oxidation observed in the kinetic analysis of the DADH reaction. 

The chromophoric pyridoxal phosphate coenzyme provides a useful spectrophotometric probe of catalytic events and of conformational changes that occur at the pyridoxal phosphate site of the P subunit and of the aiPi complex. Tryptophan synthase belongs to a class of pyridoxal phosphate enzymes that catalyze /3-replacement and / -elimination reactions.3 The reactions proceed through a series of pyridoxal phosphate-substrate intermediates (Fig. 7.6) that have characteristic spectral properties. Steady-state and rapid kinetic studies of the P subunit and of the aiPi complex in solution have demonstrated the formation and disappearance of these intermediates.73-90 Fig. 7.7 illustrates the use of rapid-scanning stopped-flow UV-visible spectroscopy to investigate the effects of single amino acid substitutions in the a subunit on the rate of reactions of L-serine at the active site of the P subunit.89 Formation of enzyme-substrate intermediates has also been observed with the 012P2 complex in the crystalline state.91 ... 

We will consider in this chapter the general processes by which enzymes achieve enhancement of reaction rates, basic chemical and enzymatic kinetics and inhibition, the roles of cofactors and coenzymes, the effects of environmental factors, the regulation of enzyme activity, and some clinical applications of enzymology. 

As shown in Figure 8.4, the synthesis of NAD from tryptophan involves the nonenzymic cyclization of aminocarhoxymuconic semialdehyde to quinolinic acid. The alternative metahoUc fate of aminocarhoxymuconic semialdehyde is decarboxylation, catalyzed hy picolinate carboxylase, leading into the oxidative branch of the pathway, and catabolism via acetyl coenzyme A. There is thus competition between an enzyme-catalyzed reaction that has hyperbolic, saturable kinetics, and a nonenzymic reaction thathas linear, first-order kinetics. 

Steady-state kinetic analysis shows that biotin-dependent reactions proceed by way of a two-site ping-pong mechanism the two-part reactions are catalyzed at distinct sites in the enzyme. These sites may be on the same or different polypeptide chains in different biotin-dependent enzymes. The e-amino linkage of lysine to the side chain of biotin in biocytin allows considerable movement of the coenzyme - the distance from C-2 of lysine to C-5 of biotin is IdA, thus allowing movement of biotin between the carboxylation and carboxyltransfer sites. 

Numerous analogs of adenosylcobalamin have been tested for their ability to replace or to inhibit the action of the coenzyme in the adenosyl-cobalamin-dependent ribonucleotide reductase reaction the enzyme from L. leichmannii has been used in most of these studies. Kinetic studies have been used in most investigations of analog-enzyme interactions and thus the interpretation of data regarding the affinity of analogs for the reductase is subject to the limitations imposed on kinetic studies of a complex reaction. 

There are reciprocal relationships between the parameters summarized above. On the one hand enzyme stability measurements strongly depend on the concentrations of substrates, coenzymes, buffers etc. in the assay. On the other hand the choice of an appropriate concentration level is a consequence of the enzyme kinetics investigated afterwards. A compromise has to be found between different optimization criteria e. g. a lower temperature leads to a reduced enzyme activity but results in a higher enzyme stability. In the example of the oxynitrilase reaction (Eq. (12)) a low pH value is a prerequisite for high enantiomeric purity of the product but lowers enzyme activity. As a consequence, only a rough optimization can be carried out at this level. 

Nicotinamide-nucleotide-linked dehydrogenases were among the earliest two-substrate enzymes to be subjected to detailed kinetic study by steady-state 1-3) and rapid reaction techniques (4), and provided much of the original stimulus for the necessary extension of kinetic theory already developed for one-substrate and hydrolytic enzymes S-8). This was partly because of the convenience and precision with which rates can be measured by means of the light absorption or fluorescence emission 9-11) of the reduced coenzymes and because of the changes of these properties which accompany the binding of reduced coenzymes to many dehydrogenases 12,13). 

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