His230 of serine hydroxymethyltransferase facilitates the proton abstraction step in catalysis

The three-dimensional structures of rabbit and human liver cytosolic serine hydroxymethyltransferase revealed that H231 interacts with the O3' of pyri doxal-5'-phosphate and other residues at the active site such as S203, K257 , H357 and R402 (numbering as per the human enzyme). This and the conserved nature of H231 in all serine hydroxymethyltransferases highlights its impo rtance in catalysis and/or maintenance of oligomeric structure of the enzym e. In an attempt to decipher the role of H230 (H231 of the human enzyme) in the catalytic mechanism and/or maintenance of oligomeric structure of shee p liver serine hydroxymethyltransferase, the residue was mutated to arginin e, phenylalanine, alanine, asparagine or tyrosine. Our results suggest that the nature of the amino acid substitution has a marked effect on the catal ytic activity of the enzyme. H230R and H230F mutant proteins were completel y inactive, dimeric and did not bind pyridoxal-5'-phosphate. On the other h and, mutation to alanine and asparagine retained the oligomeric structure a nd ability to bind pyridoxal-5'-phosphate. These mutants had only 2-3% cata lytic activity. The side reactions like transamination and 5,6,7,8-tetrahyd rofolate independent aldol cleavage were much more severely affected. They were able to form the external aldimine with glycine and serine but the qui nonoid intermediate was not observed upon the addition of 5,6,7,8-tetrahydr ofolate. Mutation to tyrosine did not affect the oligomeric structure and p yridoxal-5'-phosphate binding. The H230Y enzyme was 10% active and showed a correspondingly lower amount of quinonoid intermediate. The k(cat)/K-m val ues for L-serine and L-allothreonine were 10-fold and 174-fold less for thi s mutant enzyme compared to the wild-type protein. These results suggest th at H230 is involved in the step prior to the formation of the quinonoid int ermediate, possibly in orienting the pyridine ring of the cofactor, in orde r to facilitate effective proton abstraction.