Comparative study of the catalytic domain of phosphorylating glyceraldehyde-3-phosphate dehydrogenases from bacteria and archaea via essential cysteine probes and site-directed mutagenesis.
Phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GraP-DH) catalyzes the oxidative phosphorylation of D-glyceraldehyde-3-phosphate to form 1.3-diphosphoglycerate. The currently accepted mechanism involves an oxidoreduction step followed by a phosphorylation. Two essential aminoacids, Cys149 and His176 are involved in the chemical mechanism of bacterial and eukaryotic GraP-DHs. Roles have been assigned to the His176 as (a) a chemical activator for enhancing the reactivity of Cys149, (b) a stabilizator of the tetrahedral transition states, and (c) a base catalyst facilitating hydride transfer towards NAD. In a previous study carried out on Escherichia coli GraP-DH [Soukri, A., Mougin, A., Corbier, C., Wonacott, A. J., Branlant, C. & Branlant, G. (1989) Biochemistry, 28, 2586-2592], the role of His176 as an activator of the reactivity of Cys149 was studied. Here, we further investigated the role of the His residue in the chemical mechanism of phosphorylating GraP-DH from E. coli and Bacillus stearothermophilus. The chemical reactivity of Cys149 in the His176Asn mutant was reinvestigated. At neutral pH, its reactivity was shown to be at least as high as that observed in the Cys-/His+ ion pair present in the wild type. No pre-steady state burst of NADH was found with the His176Asn mutant in contrast to what is observed for the wild type, and a primary isotope effect was observed when D-[1-2H]glyceraldehyde-3-phosphate was used as the substrate. Therefore, the major role of the His176 in the catalytic mechanism under physiological conditions is not to activate the nucleophilicity of Cys149 but first to facilitate the hydride transfer. These results hypothesized that a phosphorylating GraP-DH possessing a different protein environment competent to increase the nucleophilic character of the essential Cys residue and to favor the hydride transfer in place of His, could be enzymically efficient. This is most likely the case for archaeal Methanothermus fervidus GraP-DH which shares less than 15% amino-acid identity with the bacterial or eukaryotic counterparts. No Cys-/His+ ion pair was detectable. Only one thiolate entity was observed with an apparent pKa of 6.2. This result was confirmed by the fact that none of the mutations of the five invariant His changed the catalytic efficiency.
Talfournier
F
F
UMR 7567 CNRS-UHP Maturation des ARN et Enzymologie Moléculaire, Vandoeuvre-les-Nancy, France.
Colloc'h
N
N
Mornon
J P
JP
Branlant
G
G
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001051:Apoenzymes / Q000737:chemistry / Q000378:metabolism; D001105:Archaea / Q000201:enzymology*; D001665:Binding Sites; D003545:Cysteine; D004926:Escherichia coli / Q000201:enzymology*; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D001051:Apoenzymes; D011994:Recombinant Proteins; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D003545:Cysteine