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His230 of serine hydroxymethyltransferase facilitates the proton abstraction step in catalysis.
Eur J Biochem. 2000 Mar; 267(5):1441-6.EJ

Abstract

The three-dimensional structures of rabbit and human liver cytosolic serine hydroxymethyltransferase revealed that H231 interacts with the O3' of pyridoxal-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 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 pyridoxal-5'-phosphate. On the other hand, mutation to alanine and asparagine retained the oligomeric structure and ability to bind pyridoxal-5'-phosphate. These mutants had only 2-3% catalytic activity. The side reactions like transamination and 5,6,7, 8-tetrahydrofolate 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 5,6,7,8-tetrahydrofolate. Mutation to tyrosine did not affect the oligomeric structure and pyridoxal-5'-phosphate binding. The H230Y enzyme was 10% active and showed a correspondingly lower amount of quinonoid intermediate. The kcat / Km values for L-serine and Lallothreonine were 10-fold and 174-fold less for this mutant enzyme compared to the wild-type 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.

Authors+Show Affiliations

Department of Biochemistry, Indian Institute of Science, Bangalore, India.No affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

10691982

Citation

Talwar, R, et al. "His230 of Serine Hydroxymethyltransferase Facilitates the Proton Abstraction Step in Catalysis." European Journal of Biochemistry, vol. 267, no. 5, 2000, pp. 1441-6.
Talwar R, Jagath JR, Rao NA, et al. His230 of serine hydroxymethyltransferase facilitates the proton abstraction step in catalysis. Eur J Biochem. 2000;267(5):1441-6.
Talwar, R., Jagath, J. R., Rao, N. A., & Savithri, H. S. (2000). His230 of serine hydroxymethyltransferase facilitates the proton abstraction step in catalysis. European Journal of Biochemistry, 267(5), 1441-6.
Talwar R, et al. His230 of Serine Hydroxymethyltransferase Facilitates the Proton Abstraction Step in Catalysis. Eur J Biochem. 2000;267(5):1441-6. PubMed PMID: 10691982.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - His230 of serine hydroxymethyltransferase facilitates the proton abstraction step in catalysis. AU - Talwar,R, AU - Jagath,J R, AU - Rao,N A, AU - Savithri,H S, PY - 2000/2/26/pubmed PY - 2000/4/15/medline PY - 2000/2/26/entrez SP - 1441 EP - 6 JF - European journal of biochemistry JO - Eur J Biochem VL - 267 IS - 5 N2 - The three-dimensional structures of rabbit and human liver cytosolic serine hydroxymethyltransferase revealed that H231 interacts with the O3' of pyridoxal-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 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 pyridoxal-5'-phosphate. On the other hand, mutation to alanine and asparagine retained the oligomeric structure and ability to bind pyridoxal-5'-phosphate. These mutants had only 2-3% catalytic activity. The side reactions like transamination and 5,6,7, 8-tetrahydrofolate 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 5,6,7,8-tetrahydrofolate. Mutation to tyrosine did not affect the oligomeric structure and pyridoxal-5'-phosphate binding. The H230Y enzyme was 10% active and showed a correspondingly lower amount of quinonoid intermediate. The kcat / Km values for L-serine and Lallothreonine were 10-fold and 174-fold less for this mutant enzyme compared to the wild-type 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. SN - 0014-2956 UR - https://www.unboundmedicine.com/medline/citation/10691982/His230_of_serine_hydroxymethyltransferase_facilitates_the_proton_abstraction_step_in_catalysis_ L2 - https://onlinelibrary.wiley.com/resolve/openurl?genre=article&sid=nlm:pubmed&issn=0014-2956&date=2000&volume=267&issue=5&spage=1441 DB - PRIME DP - Unbound Medicine ER -