The three-dimensional structures of human and rabbit liver cytosolic recombinant serine hydroxymethyltransferases (heSHMT and reSHMT) revealed that E75 and Y83 (numbering according to heSHMT)are probable candidates for proton abstraction and Ca-Cb bond cleavage in the reaction catalyzed by serine hydroxymethyltransferases sequenced to date. In an attempt to decipher the role of these residues in sheep liver cytosolic recombinant serine hydroxymethyltransferase (seSHMT), E74 (correspoding reisdue is E75 in heSHMT) was mutated to Q and K, and Y82 (corresponding residue is Y83 in heSHMT) was mutated to F. The speicfic activities using serine as the substrate for the E74Q and E74K mutant enzymes were drastically reduced. These mutant enzymes catalyzed the rates comparable with wild-type enzyme, suggesting that E74 was not involved directly in the proton abstraction step of caatalysis, as predicted earlier from crystal structures of heSHMT and reSHMT. There was no change in the apparent Tm valueof seSHMT was enhanced by 10 0C. Differential scanning calorimetric data and proteolytic digestion patterns in the presence of L-serine showed that E74Q was different to seSHMT. These results indicated that E74 might be required for the conformational change involved in hemiacetal formation following Ca-Cb bond cleavage of L-Serine and mutation of this residue to F could lead to a rapid release of HCHO. However, the Y82F mutant had only 5% of the activity and failed to form a quinonoid intermediate, suggesting that this residue is not involved in the formation of the hemiacetal intermediate, but might be involved indirectly in the abstraction of the proton and in stabilizing the quinonoid intermediate.