Role of glutamate-268 in the catalytic mechanism of nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase from Streptococcus mutans

Nonphosphorylating nicotinamide adenine dinucleotide (phosphate)- [NAD(P)-] dependent aldehyde dehydrogenases share a number of conserved amino acid r esidues, several of which are directly implicated in catalysis. In the pres ent study, the role of Glu-268 from nonphosphorylating glyceraldehyde 3-pho sphate dehydrogenase (GAPN) from Streptococcus mutans was investigated. Its substitution by Ala resulted in a k(cat) decrease by 3 orders of magnitude . Pre-steady-state analysis showed that, for both the wild-type and E268A G APNs, the rate-limiting step of the reaction is associated with deacylation . The pH dependence of the rate of acylation of wild-type GAPN is character ized by the contributions of distinct enzyme protonic species with two pK(a )s of 6.2 and 7.5. Substitution of Glu-268 by Ala resulted in a monosigmoid al pH dependence of the rate constant of acylation with a pK(a) of 6.2, whi ch suggested the assignment of pK(a) 7.5 to Glu-268. Moreover, the E268A su bstitution did not significantly affect the efficiency of acylation of GAPN , showing that Glu-268 is not critically involved in the acylation, which i ncludes Cys-302 nucleophilic activation and hydride transfer. On the contra ry, the drastic decrease of the steady-state rate constant for the E268A GA PN demonstrated the essential role of Glu-268 in the deacylation. At basic pH, the solvent isotope effect of 2.3, characterized by a unique pK(a) of 7 .7, and the linearity of the proton inventory showed that the rate-limiting process for deacylation is associated with the hydrolysis step and suggest ed that the glutamate form of Glu-268 acts as a base catalyst in this proce ss. Surprisingly, the double-sigmoidal form of the pH-steady-state rate con stant profile, characterized by pK(a) values of 6.1 and 7.4. revealed the h igh efficiency of the deacylation even at pH lower than 7.4. Therefore, we propose that the major role of Glu-268 is to promote deacylation through ac tivation and orientation of the attacking water molecule, and in addition t o act as a base catalyst at basic pH. From these results in relation to tho se recently described [Marchal, S., and Branlant, G. (1999) Biochemistry 38 , 12950-12958], a scenario for the chemical catalysis of GAPN is proposed.