The role of Glu74 and Tyr82 in the reaction catalyzed by sheep liver cytosolic serine hydroxymethyltransferase

The three-dimensional structures of human and rabbit liver cytosolic recomb inant serine hydroxymethyltransferases (hcSHMT and rcSHMT) revealed that E7 5 and Y83 (numbering according to hcSHMT) are probable candidates for proto n abstraction and Ca-CP bond cleavage in the reaction catalyzed by serine h ydroxymethyltransferase. Both these residues are completely conserved in al l serine hydroxymethyltransferases sequenced to date. In an attempt to deci pher the role of these residues in sheep liver cytosolic recombinant serine hydroxymethyltransferase (scSHMT), E74 (corresponding residue is E75 in hc SHMT) was mutated to Q and K, and Y82 (corresponding residue is Y83 in hcSH MT) was mutated to F. The specific activities using serine as the substrate for the E74Q and E74K mutant enzymes were drastically reduced. These mutan t enzymes catalyzed the transamination of D-alanine and 5,6,7,8-tetrahydrof olate independent retroaldol cleavage of L-allo threonine at rates comparab le with wild-type enzyme, suggesting that E74 was not involved directly in the proton abstraction step of catalysis, as predicted earlier from crystal structures of hcSHMT and rcSHMT. There was no change in the apparent T-m v alue of E74Q upon the addition of L-serine, whereas the apparent T-m value of scSHMT was enhanced by 10 degrees C. Differential scanning calorimetric data and proteolytic digestion patterns in the presence of L-serine showed that E74Q was different to scSHMT. These results indicated that E74 might b e required for the conformational change involved in reaction specificity. It was predicted from the crystal structures of hcSHMT and rcSHMT that Y82 was involved in hemiacetal formation following C alpha-C beta bond cleavage of L-serine and mutation of this residue to F could lead to a rapid releas e 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 invol ved in the formation of the hemiacetal intermediate, but might be involved indirectly in the abstraction of the proton and in stabilizing the quinonoi d intermediate.