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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

Jala, Venkatakrishna Rao and Ambili, M and Prakash, V and Appaji Rao, N and Savithri, HS (2003) Disruption of distal interactions of Arg 262 and of substrate binding to Ser 52 affect catalysis of sheep liver cytosolic serine hydroxymethyltransferase. In: Indian Journal of Biochemistry & Biophysics, 40 (4). pp. 226-237.

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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 K-m, for L-Ser and a 5-fold decrease in k(cat) 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 K-m and 20-fold decrease in catalytic activity efficiency for S52C SHMT, whereas S52A SHMT retained 20% of the activity without change in K-m for serine. These results suggest that S52 affects substrate binding and catalysis.

Item Type: Journal Article
Publication: Indian Journal of Biochemistry & Biophysics
Publisher: National Institute of Science Communication and Information Resources
Additional Information: Copyright of this article belongs to National Institute of Science Communication and Information Resources.
Keywords: active site geometry;distal interactions;geminal diamine; glycine-external aldimine; pyridoxal-5 '-phosphate;serine hydroxymethyltransferase (SHMT);site-directed mutagenesis.
Department/Centre: Division of Biological Sciences > Biochemistry
Date Deposited: 18 May 2009 06:18
Last Modified: 18 May 2009 06:18
URI: http://eprints.iisc.ac.in/id/eprint/17591

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