Characterization of the Amino Acids Involved in Substrate Specificity of Nonphosphorylating Glyceraldehyde-3-Phosphate Dehydrogenase from Streptococcus mutans

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 nonphosphorylating 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 ratelimiting 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 GAPN⅐NADP⅐D-G3P.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. It belongs to the aldehyde dehydrogenase (ALDH) superfamily, which oxidizes a wide variety of aldehydes into nonactivated or SCoA-activated acidic compounds (1-3). These enzymes play parts at several levels of cellular metabolism including anabolic and catabolic pathways, detoxification processes, and embryogenesis development. Their numerous functions probably explain why most ALDHs show a wide substrate specificity, except for the CoA-dependent ALDH. In mammals, ALDH class 1 shows preference for retinal but it can also oxidize aromatic and "long-chain" aldehydes with dissimilar catalytic efficiencies, whereas ALDHs that belong ...

A Differential Redox Regulation of the Pathways Metabolizing Glyceraldehyde-3-Phosphate Tunes the Production of Reducing Power in the Cytosol of Plant Cells

“…Results on the protection by substrates agree with the catalytic mechanism described for np -Ga3PDHase, where NADP + binds first to the enzyme and induces a local structural re-arrangement making accessible and leaving well positioned the catalytic Cys residue to form a competent thiohemiacetal intermediate [18]. Although NADP + binding was found to expose the catalytic Cys residue, it seems that the conformational change could not favor the reaction of the enzyme with H 2 O 2 , giving no specific insight about the involvement of that Cys residue in the redox regulation mechanism.…”

Methylmalonate-semialdehyde Dehydrogenase from Bacillus subtilis

“…The second-order rate constant k 2 is decreased by a factor of 55, 62, and at least 512 for R124L, R301L, and R124L/R301L MSDHs, respectively. The magnitude of these decreases is 3-30-fold higher than that described for R124L GAPN (28). In the case of MSDH, the decrease of k 2 likely includes contributions from both K app and k obs (max) .…”

“…1). In GAPN from Streptococcus mutans, these Arg residues were shown to be critical in the binding of D-glyceraldehyde 3-phosphate through stabilizing interactions with the C-3 phosphate (13,28). Moreover, the The sequence alignment includes some MSDHs whose activity has been demonstrated.…”

Invariant Thr244 is essential for the efficient acylation step of the non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Streptococcus mutans

“…accessible within the active site of K178A GAPN, in contrast with what was observed for the wild-type apo form [9]. The high accessibility of Cys 302 is probably not catalytically relevant, but rather is another example of the great conformational flexibility exhibited by this residue within the active site [9,26]. The apo form of K178A GAPN incubated at 4 • C in the presence of saturating concentrations of NADP for 5 h at pH 5.5 under conditions where the conformational rearrangement of the active site was shown to occur in the apo wild-type, displayed a similar pH-k 2 profile to that described for the apo K178A GAPN with pK app values of 8.85 and 9.25 and k 2 values of 3.0 × 10 5 and 2.9 × 10 4 M −1 • s −1 respectively (curves not shown, see Table 2).…”