Kinetic studies on the inhibition of isopeptidase T by ubiquitin aldehyde.

Isopeptidase T (IPaseT) can hydrolyze isopeptide bonds of polyubiquitin (polyUb) chains, simple C-terminal derivatives of Ub, and certain peptides. We recently reported that IPaseT is regulated by ubiquitin (Ub); while submicromolar Ub activates, higher concentrations inhibit this enzyme [Stein et al. (1995) Biochemistry 34, 12616]. To explain these observations, we proposed a model for IPaseT involving two binding sites for Ub. According to the model, the two sites are adjacent to one another and are the extended active site that binds two Ub moieties of a polyUb chain. The "activation site" binds the Ub that donates Lys to the isopeptide bond. The "inhibition site" is adjacent and binds the Ub that donates the C-terminal Gly to the isopeptide bond. We now report that the interaction of IPaseT with the C-terminal aldehyde of Ub (Ub-H) is also modulated by Ub. In the absence of Ub, Ub-H inhibits IPaseT with a Ki of 2.3 nM, while at 0.6 microM Ub, where the "activation site" is occupied, Ki is less than 0.1 nM. At high Ub concentrations, where both the "activation" and "inhibition" sites are occupied, IPaseT cannot bind Ub-H. We also determined the kinetics of inhibition of IPaseT by Ub-H. In the absence of Ub, a two-step mechanism is followed. In the first step, Ub-H slowly combines with IPaseT to form a relatively weak complex (K1 = 260 nM) that slowly isomerizes to the final, stable complex that accumulates in the steady-state (k2 = 2 x 10(-3) s-1; k-2 = 0.02 x 10(-3) s-1). In contrast, Ub-activated IPaseT is inhibited by Ub-H through a three-step process. In the first step, Ub-H rapidly combines with IPaseT to form a complex (K1 = 10 nM) that slowly isomerizes to a second, more stable complex (k2 = 18 x 10(-3) s-1; k-2 = 1.5 x 10(-3) s-1). In the third step, the second complex converts to the final complex (k3 = 1.5 x 10(-3) s-1; k-3 < 0.2 x 10(-3) s-1). To unify the results of this study with our previous results on catalysis, we propose that binding of Ub either to catalytic transition states or to tetrahedral inhibition intermediates liberates more free energy than binding of Ub to the reactant state of IPaseT and that IPaseT can utilize this binding energy to stabilize both of these tetrahedral species. The overall effect is a Ub-induced increase in catalytic efficiency or inhibitory potency.

Solvent isotope effects and the nature of electrophilic catalysis in 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.

Progress in establishing the rate-limiting features and kinetic mechanism of the glyceraldehyde-3-phosphate dehydrogenase reaction.

Primary hydrogen isotope effects and steady-state kinetics have been used to study the mechanism of glyceraldehyde-3-phosphate (GAP) dehydrogenase at pH 8.6. The isotope effect determined by using GAP-1d was unity and independent of arsenate (used as the acyl acceptor) and NAD+ concentrations when the aldehyde substrate was at saturating concentrations. At low GAP concentrations (apparent V/K conditions), the primary hydrogen isotope effect (H/D) was in the range of 1.40-1.52 and independent of arsenate and NAD+ concentrations. Apparent V/K for NAD+ was independent of GAP concentration, and apparent V/K for GAP was independent of NAD+ concentration. The dependence of apparent V/K for GAP on arsenate concentration was more complex but extrapolated to nonzero V/K at the zero-arsenate intercept. These observations are consistent with the general features of the Segal and Boyer (1953) mechanism for the reaction.

Chiral instability at sulfur of S-adenosylmethionine.

S-Adenosylmethionine, generated enzymically in chirally pure form (S configuration at sulfur), undergoes simultaneous irreversible conversion to 5'-deoxy-5'-(methylthio)adenosine and homoserine with a rate constant of 6 X 10(-6) s-1 at pH 7.5 and 37 degrees C and reversible conversion to an enzymically inactive stereoisomer (R configuration at sulfur) with a forward rate constant of 8 X 10(-6) s-1 at pH 7.5 and 37 degrees C. These forms of instability require small turnover times and/or stabilization through macromolecular binding for S-adenosylmethionine, if organisms that utilize it are to avoid losses of metabolic energy.