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 recombinant serine hydroxymethyltransferases
(hcSHMT and rcSHMT) revealed that E75 and Y83 (numbering according to hcSHMT) are probable
candidates for proton abstraction and Ca-Cb bond cleavage in the reaction catalyzed by serine hydroxymethyltransferase.
Both these residues are completely conserved in all serine hydroxymethyltransferases sequenced to
date. In an attempt to decipher the role of these residues in sheep liver cytosolic recombinant serine
hydroxymethyltransferase (scSHMT), E74 (corresponding residue is E75 in hcSHMT) was mutated to Q and K,
and Y82 (corresponding residue is Y83 in hcSHMT) was mutated to F. The specific activities using serine as the
substrate for the E74Q and E74K mutant enzymes were drastically reduced. These mutant enzymes catalyzed the
transamination of d-alanine and 5,6,7,8-tetrahydrofolate independent retroaldol cleavage of l-allo threonine at
rates comparable 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 Tm value of E74Q upon the addition of l-serine, whereas the apparent Tm value of scSHMT was
enhanced by 10 8C. 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 be 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 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.