Abstract
In order to address the molecular basis of the specificity of aldehyde dehydrogenase for aldehyde substrates, enzymatic characterization of the glyceraldehyde 3-phosphate (G3P) binding site of non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN) from Streptococcus mutans has been undertaken. In this work, residues Arg-124, Tyr-170, Arg-301, and Arg-459 were changed by site-directed mutagenesis and the catalytic properties of GAPN mutants investigated. Changing Tyr-170 into phenylalanine induces no major effect on k(cat) and K(m) for d-G3P in both acylation and deacylation steps. Substitutions of Arg-124 and Arg-301 by leucine and Arg-459 by isoleucine led to distinct effects on K(m), on k(cat), or on both. The rate-limiting step of the R124L GAPN remains deacylation. Pre-steady-state analysis and substrate isotope measurements show that hydride transfer remains rate-determining in acylation. Only the apparent affinity for d-G3P is decreased in both acylation and deacylation steps. Substitution of Arg-459 by isoleucine leads to a drastic effect on the catalytic efficiency by a factor of 10(5). With this R459L GAPN, the rate-limiting step is prior to hydride transfer, and the K(m) of d-G3P is increased by at least 2 orders of magnitude. Binding of NADP leads to a time-dependent formation of a charge transfer transition at 333 nm between the pyridinium ring of NADP and the thiolate of Cys-302, which is not observed with the holo-wild type. Accessibility of Cys-302 is shown to be strongly decreased within the holostructure. The substitution of Arg-301 by leucine leads to an even more drastic effect with a change of the rate-limiting step similar to that observed for R459I GAPN. Taking into account the three-dimensional structure of GAPN from S. mutans and the data of the present study, it is proposed that 1) Tyr-170 is not essential for the catalytic event, 2) Arg-124 is only involved in stabilizing d-G3P binding via an interaction with the C-3 phosphate, and 3) Arg-301 and Arg-459 participate not only in d-G3P binding via interaction with C-3 phosphate but also in positioning efficiently d-G3P relative to Cys-302 within the ternary complex GAPN.NADP.d-G3P.
Highlights
Nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN)1 catalyzes the irreversible oxidation of D-glyceraldehyde 3-phosphate (D-G3P) into 3-phosphoglycerate in the presence of NADP via a two-step chemical mechanism
Taking into account the three-dimensional structure of GAPN from S. mutans and the data of the present study, it is proposed that 1) Tyr-170 is not essential for the catalytic event, 2) Arg-124 is only involved in stabilizing D-G3P binding via an interaction with the C-3 phosphate, and 3) Arg-301 and Arg-459 participate in D-G3P binding via interaction with C-3 phosphate and in positioning efficiently D-G3P relative to Cys-302 within the ternary complex GAPN1⁄7NADP1⁄7D-G3P
The fact that adding D-G3P increases the rate of the reorganization by a factor of at least 105-fold [14]6 indicates that D-G3P binding to the binary complex GAPN1⁄7NADP strongly favors the active site reorganization
Summary
Materials—Production and purification procedures of wild-type and mutated GAPNs were carried out as previously described [14]. One syringe was filled with mutant enzymes (16 N after mixing), and the other one contained NADP and D-G3P (1 mM after mixing) Under these conditions, the acylation step was shown to be rate-limiting for only the R301L and R459I mutant GAPN proteins (see “Results”). The second-order rate constant kac/Km, which corresponds to the efficiency of the acylation step, was calculated at each pH, dividing kac by the concentration of D-G3P and was fitted to Equation 1 or 2 to determine the best fit pKa values,. In the case of R124L and Y170F mutant GAPN proteins, kinetics of the acylation step were carried out on a stopped-flow apparatus at 25 °C under similar conditions to those already described for the wild type [16]. The second-order kinetic constants k2 were calculated dividing the kobs values by the concentration of 2PDS and were fitted to Equation 3 to determine the best-fit pKapp values. Pseudo-first-order rate constants kobs were determined at each pH by fitting the absorbance A at 333 nm versus time t to an equation for a single exponential profile and were fitted to Equation 6 to determine the best-fit pKapp values, where kЈ and kЉ represent two different rate constants for the thiolate form
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