Characterization of the amino acids involved in substrate specificity of nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase from Streptococcus mutans.
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.
Marchal
Stephane
S
Laboratoire de Maturation des ARN et Enzymologie Moléculaire, UMR CNRS-UHP 7567, Université Henri Poincaré Nancy 1, Faculté des Sciences, BP 239, F-54506 Vandoeuvre-lès-Nancy Cédex, France.
Branlant
Guy
G
D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry / Q000378:metabolism; D002384:Catalysis; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D010649:Phenylalanine / Q000737:chemistry; D010710:Phosphates / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013295:Streptococcus mutans / Q000201:enzymology* / Q000378:metabolism; D013379:Substrate Specificity; D013997:Time Factors; D014443:Tyrosine / Q000737:chemistry / Q000378:metabolism
D010710:Phosphates; D014443:Tyrosine; D010649:Phenylalanine; D001120:Arginine; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases