K. Barbara Schowen

[29] Solvent isotope effects on enzyme systems

Publisher Summary This chapter discusses the solvent isotope effects on enzyme systems. The introduction of deuterium in place of protium in the hydrogenic sites of water, and its consequent exchange into some positions of enzymes and substrates produces solvent isotope effects on the kinetic and equilibrium constants associated with the enzymic reaction. These effects, usually expressed as ratios of the appropriate constants in the two isotopic solvents HOH and DOD, are useful in the study of reaction mechanism. The chapter provides (a) the physicochemical background for the use of solvent isotope effects in biochemical studies, with fairly complete derivations of the requisite algebraic expressions, an account of underlying assumptions, and a review of pertinent experimental and theoretical information. (b) to outline workable procedures for carrying out experiments in this area; and (c) to present the apparatus for interpretation of the results.

The Use of Isotope Effects to Elucidate Enzyme Mechanisms

The changes in molecular structure that occur in the course of enzymecatalyzed chemical reactions (enzyme mechanisms) can be investigated by measuring relative reaction rates with isotopic molecules. In general, the aim of research on enzyme mechanisms is to reveal how enzymes have altered the mechanisms of the inherently slow chemical processes that dominate in biology, in order to make them rapid. For this purpose, one often determines the reaction mechanism in the absence of the enzyme and then in its presence and under its influence. By comparison, one develops a picture of how the enzyme intervenes to change and to facilitate the mechanism. In addition, one may subject the enzymic reaction itself to quite detailed study in order to learn with precision all that is involved in the action of the enzyme. Enzyme mechanisms are mainly of fundamental significance, but they also have some practical importance in pharmacology and medicine. A large number of drugs are enzyme inhibitors, and current approaches to the rational design of drugs rely heavily on information about the mechanism of action of the target enzyme system (Gandour and Schowen 1978, p. 555-591).

Hydrogen Tunneling in Glucose Oxidation by the Archaeon Thermoplasma acidophilum

Abstract The thermophilic archaeon Thermoplasma acidophilum, with an optimal growth temperature in the region of 60 °C, has evolved a D-glucose dehydrogenase, dependent on NADP+ and accepting only the β-anomer of D-glucose, that exhibits a temperature dependence of the rate constants kcat/Kmβ for 1-h-β-D-glucose and 1-d-β-D-glucose that indicate two modes of quantum tunneling in the hydride-transfer reaction from substrate to NADP+. Near the optimal temperature for the organism, tunneling seems to occur in a prepared configuration that has most logically been designed by molecular evolution. At lower temperatures, a discontinuity in the temperature dependence of the catalytic rate constant is observed and is thought to arise from a protein structural transition. Below the transition temperature, tunneling appears to occur by a mechanism involving sequential configurational searches for a tunneling state, as is more commonly observed in non-enzymic reactions.

The linkage of catalysis and regulation in enzyme action: oxidative diversion in the hysteretically regulated yeast pyruvate decarboxylase

The reaction catalyzed by the thiamin-diphosphate-dependent yeast pyruvate decarboxylase, which is hysteretically regulated by pyruvate, undergoes paracatalytic oxidative diversion by 2,6-dichlorophenolindophenol, which traps a carbanionic intermediate and diverts the product from acetaldehyde to acetate (Christen, P. Meth. Enzymol. 1977, 46, 48). This reaction is now shown to exhibit an oxidant on-rate constant somewhat faster than that for pyruvate in the normal catalytic cycle and a product off-rate constant about 60-fold smaller than that for acetaldehyde. Both on-rates and off-rates exhibit an inverse solvent isotope effect of 1.5-2, observed in normal catalysis as a signal of sulfhydryl addition to the keto group of pyruvate at the allosteric regulatory site. The findings are consistent with a model for regulation in which the sulfhydryl-addition process mediates access to a fully catalytically competent active site, the oxidative-diversion reaction being forced to make use of the normal entry exit machinery.

Solvent isotope effects and the nature of electrophilic catalysis m the action of the lactate dehydrogenase of bacillus stearothermophilus

Deuterium oxide at atom fractions of deuterium from 0.0 to 0.97 has an effect of less than 20% on the kinetic term kcat/KmB (believed to reflect the transition state for the hydride-transfer step) for the reduction of pyruvic acid by NADH at 55 degrees C, with catalysis by the tetrameric form of the lactate dehydrogenase of Bacillus stearothermophilus. This observation suggests that the hydride-transfer event is not assisted by protonic bridging to the carbonyl group being reduced. The results are consistent with protonic bridging only if an opposing isotope effect is present, for example from a generalized conformation or solvation change. The results are consistent with other forms of electrophilic catalysis.

Isotope Effects and Temperature Dependences in the Action of the Glucose Dehydrogenase of the Mesophilic Bacterium Bacillus megaterium.

The glucose dehydrogenase of the mesophilic bacterium Bacillus megaterium (optimal growth around 35 °C) exhibits non-linear Eyring temperature dependences from 25 to 55 °C in its catalysis of the oxidation by hydride-transfer to NAD+ of the β-anomers of 1-h-D-glucose and 1-d-D-glucose (rate constant kcat/KMβ). A break around 300K separates a high-T region from a low-T region. In the high-T region, isotopic enthalpies of activation within a considerable experimental error are equal to zero. In the low-T region, the enthalpies of activation are roughly equal for the isotopic substrates but are different from zero. An alternative treatment with Eyring plots taken as effectively linear produces enthalpies of activation having the unusual feature of being larger for the H-substrate (26 kJ/mol) than for the D-substrate (21 kJ/mol). Compensation of the enthalpic effect by a more positive entropy for the H-substrate then reproduces the isotope effects. For oxidation by NADP+ of the same pair of isotopic glucose substrates, catalysis by the glucose dehydrogenase of Thermoplasma acidophilum, a thermophilic archaeon, leads to temperature dependences characterized by a high-T region and a low-T region separated by a gentle thermal transition (K. Anandarajah, K.B. Schowen, and R.L. Schowen, Z. phys. Chem. 2008, 222, 1333-1347). Tentative approaches to a mechanistic interpretation of both cases rely on models featuring configurational searches of the enzyme for tunneling states, followed by hydrogen-transfer tunneling, although explanations can be constructed also on the basis of simple transition-state stabilization without tunnelling.