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

Krishna Rao, J. V. ; Prakash, V. ; Appaji Rao, N. ; Savithri, H. S. (2000) The role of Glu74 and Tyr82 in the reaction catalyzed by sheep liver cytosolic serine hydroxymethyltransferase European Journal of Biochemistry, 267 (19). pp. 5967-5976. ISSN 0014-2956

PDF - Publisher Version

Official URL:

Related URL:


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 Cα-Cβ 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 lallo 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°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 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 Cα-Cβ 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.

Item Type:Article
Source:Copyright of this article belongs to John Wiley and Sons, Inc.
Keywords:Active Site Residues; Conformational Change; Proton Abstraction; Serine Hydroxymethyltransferase; Spectral Intermediates
ID Code:21244
Deposited On:20 Nov 2010 13:16
Last Modified:17 May 2016 05:27

Repository Staff Only: item control page