The active site of phosphorylating glyceraldehyde-3-phosphate dehydrogenase is not designed to increase the nucleophilicity of a serine residue

Changing a catalytic cysteine into a serine, and vice versa, generally lead s to a dramatic decrease in enzymatic efficiency. Except a study done on th iol subtilisin, no extensive study was carried out for determining whether the decrease in activity is due to a low nucleophilicity of the introduced amino acid. In the present study, Cys149 of glyceraldehyde-3-phosphate dehy drogenase from Bacillus stearothermophilus was converted into a Ser residue . This leads to a drastic reduction of the k(cat) value. The rate-limiting step occurs before the hydride transfer step. Selective, but slow, inactiva tion is observed with specific, structurally different, inhibitors of serin e protease. The esterolytic activity of serine mutant towards activated est ers is also strongly decreased. The rate-limiting step of the esterase reac tion also shifts from deacylation in the wild type to acylation in the muta nt. Altogether, these results strongly suggest that the low catalytic effic iency of the Ser mutant is due to a poor nucleophilicity of the hydroxyl se rine group within the active site of the enzyme. The fact that (1) the apo --> hole transition does not change esterolytic and inactivating efficienci es, and (2) Ser149 Asn176 double mutant exhibits the same chemical reactivi ty and esterolytic catalytic efficiency compared to the Ser149 single mutan t indicates that the serine residue is not subject to His176 general base c atalysis. A linear relationship between the catalytic dehydrogenase rate, t he k(cat)/K-M for esterolysis, and the concentration of OH- is observed, th us supporting the alcoholate entity as the attacking reactive species, Coll ectively this study shows that the active site environment of GAPDH is not adapted to increase the nucleophilicity of a serine residue. This is discus sed in relation to what is known about Ser and Cys protease active sites. ( C) 1999 Academic Press.