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Disruption of distal interactions of Arg 262 and of substrate binding to Ser 52 affect catalysis of sheep liver cytosolic serine hydroxymethyltransferase.
Indian J Biochem Biophys. 2003 Aug; 40(4):226-37.IJ

Abstract

The crystal structure of human liver cytosolic recombinant serine hydroxymethyltransferase (hcSHMT) suggested that Ser53 and Arg 263 could participate in the reaction catalyzed by SHMT. The mutation of Arg262 (corresponding to Arg263 in hcSHMT) to "A" in sheep liver cytosolic SHMT (scSHMT) resulted in a 5-fold increase in Km for L-Ser and a 5-fold decrease in kcat compared to scSHMT. Further, in R262A SHMT-glycine complex, the peak at 343 nm (geminal diamine) was more pronounced, compared to wild-type enzyme. Stopped-flow studies showed that the rate constant for the formation of glycine-geminal diamine for R262A SHMT was also decreased. The rate of reaction, concentration of spectral intermediates, fluorescence excitation maximum of glycine geminal diamine and interaction with methoxyamine were altered in R262A SHMT. Although Arg263 in hcSHMT is located outside the PLP binding pocket, it positions Tyr73 for interaction with PLP, by forked H-bonding with the carbonyl groups of main chain residues, Asn71 and Lys72 of the other subunit of the tight dimer. Mutation of Arg262 to Ala and the consequent alteration in orientation of PLP leads to decreased catalytic efficiency. Ser53 (in hcSHMT) is in hydrogen bonding distance to one of the carboxylate oxygens of the amino acid substrate, which also interacts with Tyr83 and Arg402. Replacement of Ser53 with Cys (using 'O' software program) in the structure of hcSHMT resulted in disruption of these interactions, whereas replacement with Ala (S53A) only weakened the substrate interactions. There was a 10-fold increase in Km and 20-fold decrease in catalytic activity efficiency for S52C SHMT, whereas S52A SHMT retained 20% of the activity without change in Km for serine. These results suggest that S52 affects substrate binding and catalysis.

Authors+Show Affiliations

Department of Biochemistry, Indian Institute of Science, Bangalore 560 012, India.No affiliation info availableNo 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

22900314

Citation

Jala, Venkatakrishna Rao, et al. "Disruption of Distal Interactions of Arg 262 and of Substrate Binding to Ser 52 Affect Catalysis of Sheep Liver Cytosolic Serine Hydroxymethyltransferase." Indian Journal of Biochemistry & Biophysics, vol. 40, no. 4, 2003, pp. 226-37.
Jala VR, Ambili M, Prakash V, et al. Disruption of distal interactions of Arg 262 and of substrate binding to Ser 52 affect catalysis of sheep liver cytosolic serine hydroxymethyltransferase. Indian J Biochem Biophys. 2003;40(4):226-37.
Jala, V. R., Ambili, M., Prakash, V., Rao, N. A., & Savithri, H. S. (2003). Disruption of distal interactions of Arg 262 and of substrate binding to Ser 52 affect catalysis of sheep liver cytosolic serine hydroxymethyltransferase. Indian Journal of Biochemistry & Biophysics, 40(4), 226-37.
Jala VR, et al. Disruption of Distal Interactions of Arg 262 and of Substrate Binding to Ser 52 Affect Catalysis of Sheep Liver Cytosolic Serine Hydroxymethyltransferase. Indian J Biochem Biophys. 2003;40(4):226-37. PubMed PMID: 22900314.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Disruption of distal interactions of Arg 262 and of substrate binding to Ser 52 affect catalysis of sheep liver cytosolic serine hydroxymethyltransferase. AU - Jala,Venkatakrishna Rao, AU - Ambili,M, AU - Prakash,V, AU - Rao,N Appaji, AU - Savithri,H S, PY - 2012/8/21/entrez PY - 2003/8/1/pubmed PY - 2012/9/12/medline SP - 226 EP - 37 JF - Indian journal of biochemistry & biophysics JO - Indian J. Biochem. Biophys. VL - 40 IS - 4 N2 - The crystal structure of human liver cytosolic recombinant serine hydroxymethyltransferase (hcSHMT) suggested that Ser53 and Arg 263 could participate in the reaction catalyzed by SHMT. The mutation of Arg262 (corresponding to Arg263 in hcSHMT) to "A" in sheep liver cytosolic SHMT (scSHMT) resulted in a 5-fold increase in Km for L-Ser and a 5-fold decrease in kcat compared to scSHMT. Further, in R262A SHMT-glycine complex, the peak at 343 nm (geminal diamine) was more pronounced, compared to wild-type enzyme. Stopped-flow studies showed that the rate constant for the formation of glycine-geminal diamine for R262A SHMT was also decreased. The rate of reaction, concentration of spectral intermediates, fluorescence excitation maximum of glycine geminal diamine and interaction with methoxyamine were altered in R262A SHMT. Although Arg263 in hcSHMT is located outside the PLP binding pocket, it positions Tyr73 for interaction with PLP, by forked H-bonding with the carbonyl groups of main chain residues, Asn71 and Lys72 of the other subunit of the tight dimer. Mutation of Arg262 to Ala and the consequent alteration in orientation of PLP leads to decreased catalytic efficiency. Ser53 (in hcSHMT) is in hydrogen bonding distance to one of the carboxylate oxygens of the amino acid substrate, which also interacts with Tyr83 and Arg402. Replacement of Ser53 with Cys (using 'O' software program) in the structure of hcSHMT resulted in disruption of these interactions, whereas replacement with Ala (S53A) only weakened the substrate interactions. There was a 10-fold increase in Km and 20-fold decrease in catalytic activity efficiency for S52C SHMT, whereas S52A SHMT retained 20% of the activity without change in Km for serine. These results suggest that S52 affects substrate binding and catalysis. SN - 0301-1208 UR - https://www.unboundmedicine.com/medline/citation/22900314/Disruption_of_distal_interactions_of_Arg_262_and_of_substrate_binding_to_Ser_52_affect_catalysis_of_sheep_liver_cytosolic_serine_hydroxymethyltransferase_ DB - PRIME DP - Unbound Medicine ER -