The reactions of D-glyceraldehyde 3-phosphate with thiols and the holoenzyme of D-glyceraldehyde 3-phosphate dehydrogenase and of inorganic phosphate with the acyl-holoenzyme

Abstract

D-Glyceraldehyde 3-phosphate forms adducts with thiols. These adducts, which are presumed to be hemithioacetals, equilibrate rapidly with the unhydrated form of the aldehyde, which is the subtrate for D-glyceraldehyde 3-phosphate dehydrogenase. The adduct provides a substrate buffer system whereby a constant low free aldehyde concentration can be maintained during the oxidation of aldehyde by the enzyme and NAD+. With this system, the kinetics of the association of the aldehyde with the enzyme were examined. The rate profile for this reaction is a single exponential process, showing that all four active sites of the enzyme have equivalent and independent reactivity towards the aldehyde, with an apparent second-order rate constant of 5 X 10(7)M-1-S-1 at pH8.0 and 21 degrees C. The second-order rate constant becomes 8 X 10(7)M-1-S-1 when account is taken of the forward and reverse catalytic rate constants of the dehydrogenase. The pH-dependence of the observed rate constant is consistent with a requirement for the unprotonated form of a group of pK 6.1, which is the pK observed for second ionization of glyceraldehyde 3-phosphate. The rate of phosphorolysis of the acyl-enzyme intermediate during the steady-state oxidative phosphorylation of the aldehyde was studied, and is proportional to the total Pi concentration up to at least 1 mM-Pi at pH 7.5. The pH-dependence of the rate of NADH generation under these conditions can be explained by the rate law d[NADA]/dt = k[acy] holoenzyme][PO4(3-)-A1, where thioester bond, although kinetically indistinguishable rate equations for the reaction are possible. The rates of the phosphorolysis reaction and of the aldehyde-association reaction decrease with increasing ionic strength, suggesting that the active site of the enzyme has cationic groups which are involved in the reaction of the enzyme with anionic substrates.

Convergence of active center geometries

“…The opposite hand of C2 would result in steric hindrance between the phosphate on atom C3 and the nicotinamide ring. This analysis is supported by the observations of Armstrong and Trentham (1976) that the enzyme not only fails to oxidize the L substrate but that the latter does not even inhibit the oxidation of the D substrate.…”

Characterization of the two anion-recognition sites of glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus by site-directed mutagenesis and chemical modification

“…Thus, kz is likely to correspond to the association rate constant for free aldehyde. Assuming a 1 % free aldehyde concentration under the reaction conditions (Armstrong & Trentham, 1976), a true kz value of 6.4 X 10s M_1 s-1 can be calculated for the mutant R231G. This value is close to the upper limit for a substrate diffusion and is rather high for a substrate like G3P which should be strongly solvated on the C-3 phosphate.…”

“…As a consequence, at suitable enzyme concentrations required for the stopped-flow experiments, the rate of the reaction tends to be controlled by dehydration of the gem-diol form. The use of a "substrate buffer" system initially proposed by Armstrong and Trentham (1976) alleviates this problem, since the hemithioacetal adduct equilibrates rapidly with the unhydrated form of the aldehyde and thus maintains the concentration of free aldehyde constant during the oxidoreduction process. Using this approach, a linear variation of k0bS versus aldehyde concentration is observed, up to a concentration of 220 µ of G3P.…”

“…(b) Kinetics of Enzyme-Glyceraldehyde 3-Phosphate Association. Enzyme-substrate association was performed at high thiol concentration (/9-mercaptoethanol: 20 mM) using an approach initially developed by Armstrong and Trentham (1976). In these conditions, the observed pseudo-first-order constant of the transient process was measured for varying G3P concentrations (27.5-220 µ ) for wild-type and mutant enzymes.…”

A Proteome Reference Map of the Causative Agent of Melioidosis Burkholderia pseudomallei

“…For example, two enzyme isoforms encoded by glyceraldehyde 3-phosphate dehydrogenase (GapA) gene show a similar molecular weight, but their pIs are 4.47 and 6.70. This roughly 3-unit charge difference might be due to an intermediate transition, glyceraldehyde 3-phosphate-bound form of the enzyme localized at the acidic pI [ 16 , 17 ]. Thus, this result suggests that enzyme isoforms with charge difference might represent an intermediate of its catalytic step.…”