The role of His-134, -147, and -150 residues in subunit assembly, cofactor binding, and catalysis of sheep liver cytosolic serine hydroxymethyltransferase.

In an attempt to unravel the role of conserved histidine residues in the structure-function of sheep liver cytosolic serine hydroxymethyltransferase (SHMT), three site-specific mutants (H134N, H147N, and H150N) were constructed and expressed. H134N and H147N SHMTs had Km values for L-serine, L-allo-threonine and beta-phenylserine similar to that of wild type enzyme, although the kcat values were markedly decreased. H134N SHMT was obtained in a dimeric form with only 6% of bound pyridoxal 5'-phosphate (PLP) compared with the wild type enzyme. Increasing concentrations of PLP (up to 500 microM) enhanced the enzyme activity without changing its oligomeric structure, indicating that His-134 may be involved in dimer-dimer interactions. H147N SHMT was obtained in a tetrameric form but with very little PLP (3%) bound to it, suggesting that this residue was probably involved in cofactor binding. Unlike the wild type enzyme, the cofactor could be easily removed by dialysis from H147N SHMT, and the apoenzyme thus formed was present predominantly in the dimeric form, indicating that PLP binding is at the dimer-dimer interface. H150N SHMT was obtained in a tetrameric form with bound PLP. However, the mutant had very little enzyme activity (<2%). The kcat/Km values for L-serine, L-allo-threonine and beta-phenylserine were 80-, 56-, and 33-fold less compared with wild type enzyme. Unlike the wild type enzyme, it failed to form the characteristic quinonoid intermediate and was unable to carry out the exchange of 2-S proton from glycine in the presence of H4-folate. However, it could form an external aldimine with serine and glycine. The wild type and the mutant enzyme had similar Kd values for serine and glycine. These results suggest that His-150 may be the base that abstracts the alpha-proton of the substrate, leading to formation of the quinonoid intermediate in the reaction catalyzed by SHMT.