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The role of Glu74 and Tyr82 in the reaction catalyzed by sheep liver cytosolic serine hydroxymethyltransferase.
Eur J Biochem. 2000 Oct; 267(19):5967-76.EJ

Abstract

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 Calpha-Cbeta 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 degrees 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 Calpha-Cbeta 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.

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

10998057

Citation

Rao, J V., et al. "The Role of Glu74 and Tyr82 in the Reaction Catalyzed By Sheep Liver Cytosolic Serine Hydroxymethyltransferase." European Journal of Biochemistry, vol. 267, no. 19, 2000, pp. 5967-76.
Rao JV, Prakash V, Rao NA, et al. The role of Glu74 and Tyr82 in the reaction catalyzed by sheep liver cytosolic serine hydroxymethyltransferase. Eur J Biochem. 2000;267(19):5967-76.
Rao, J. V., Prakash, V., Rao, N. A., & 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), 5967-76.
Rao JV, et al. The Role of Glu74 and Tyr82 in the Reaction Catalyzed By Sheep Liver Cytosolic Serine Hydroxymethyltransferase. Eur J Biochem. 2000;267(19):5967-76. PubMed PMID: 10998057.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - The role of Glu74 and Tyr82 in the reaction catalyzed by sheep liver cytosolic serine hydroxymethyltransferase. AU - Rao,J V, AU - Prakash,V, AU - Rao,N A, AU - Savithri,H S, PY - 2000/9/21/pubmed PY - 2001/2/28/medline PY - 2000/9/21/entrez SP - 5967 EP - 76 JF - European journal of biochemistry JO - Eur. J. Biochem. VL - 267 IS - 19 N2 - 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 Calpha-Cbeta 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 degrees 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 Calpha-Cbeta 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. SN - 0014-2956 UR - https://www.unboundmedicine.com/medline/citation/10998057/The_role_of_Glu74_and_Tyr82_in_the_reaction_catalyzed_by_sheep_liver_cytosolic_serine_hydroxymethyltransferase_ L2 - https://onlinelibrary.wiley.com/resolve/openurl?genre=article&sid=nlm:pubmed&issn=0014-2956&date=2000&volume=267&issue=19&spage=5967 DB - PRIME DP - Unbound Medicine ER -