Jump to Main Content
His230 of serine hydroxymethyltransferase facilitates the proton abstraction step in catalysis
- Talwar, Rashmi, Jagath, Junutula R., Rao, N. Appaji, Savithri, H. S.
- European journal of biochemistry 2000 v.267 no.5 pp. 1441-1446
- active sites, alanine, amino acid substitution, arginine, asparagine, catalytic activity, enzyme activity, glycine hydroxymethyltransferase, humans, liver, mutants, phenylalanine, proteins, pyridines, rabbits, serine, sheep, transamination, tyrosine
- The threeâdimensional structures of rabbit and human liver cytosolic serine hydroxymethyltransferase revealed that H231 interacts with the O3â² of pyridoxalâ5â²âphosphate 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 importance in catalysis and/or maintenance of oligomeric structure of the enzyme. 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 sheep liver serine hydroxymethyltransferase, the residue was mutated to arginine, phenylalanine, alanine, asparagine or tyrosine. Our results suggest that the nature of the amino acid substitution has a marked effect on the catalytic activity of the enzyme. H230R and H230F mutant proteins were completely inactive, dimeric and did not bind pyridoxalâ5â²âphosphate. On the other hand, mutation to alanine and asparagine retained the oligomeric structure and ability to bind pyridoxalâ5â²âphosphate. These mutants had only 2â3% catalytic activity. The side reactions like transamination and 5,6,7,8âtetrahydrofolate independent aldol cleavage were much more severely affected. They were able to form the external aldimine with glycine and serine but the quinonoid intermediate was not observed upon the addition of 5,6,7,8âtetrahydrofolate. Mutation to tyrosine did not affect the oligomeric structure and pyridoxalâ5â²âphosphate binding. The H230Y enzyme was 10% active and showed a correspondingly lower amount of quinonoid intermediate. The kcatâ/âKm values for lâserine and lallothreonine were 10âfold and 174âfold less for this mutant enzyme compared to the wildâtype protein. These results suggest that H230 is involved in the step prior to the formation of the quinonoid intermediate, possibly in orienting the pyridine ring of the cofactor, in order to facilitate effective proton abstraction.