Kinetics of enzyme-coenzyme

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. 

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. 

Kinetic studies of reversible inhibition by substrate analogs give evidence of the mode of action of the inhibitor and the types of enzyme-inhibitor complex formed, and estimates of their dissociation constants. The complexes may be isolated and sometimes crystallized. Studies of the stabilities, optical properties, and structures of ternary complexes of enzymes, coenzymes, and substrate analog in particular, as stable models of the catalytically active ternary complexes or of the transition state for hydride transfer (61,79,109,115-117), can only be touched upon here there is direct evidence with several enzymes that the binding of coenzymes is firmer in such complexes than in their binary complexes (85,93,118), which supports the indirect, kinetic evidence already mentioned for a similar stabilization in active ternary complexes. 

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... 

Spin labeled 5 -deoxyadenosylcobinamide has been used as a cofactor for ethanolamine-ammonia-lyase and the ESR spectrum followed during catalysis (123). This spin labeled coenzyme is biologically active in this enzyme. Enzyme kinetics showed this derivative to have the same Vmax as the cofactor 5 -deoxyadenosylcobinamide, but it has a higher Km value of 5.1 X 10-6 M compared to 4.6 X 10-6 for 5 -deoxyadenosylcobinamide (123). When the spin labeled coenzyme was incubated with apoenzyme to give the enzyme-coenzyme complex, the nitroxide ESR spectrum resembled that of free spin label but the lines are broadened significantly. 

Bertrand, in 1897, observed that certain enzymes required dialysable substances to exert catalytic activity. He named these substances coenzymes . Sorensen pointed out the dependence of enzyme activity on pH in 1909 (Sumner and Somers, 1953). An important step entering physico-chemistry, and hence extending the theoretical basis of enzymology, were the kinetic investigations and their interpretation by Michaelis and Menten. They postulated that enzymatic action is due to the formation of an intermediate compound between enzyme and substrate, and they presented a mathematical form still used today (Sumner and Myrback, 1950). 

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. 

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. 

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