5bir

From Proteopedia

Revision as of 16:44, 5 November 2007

DISECTING HISTIDINE INTERACTIONS IN RIBONUCLEASE T1 USING ASN AND GLN MUTATIONS

Overview

His92 of Ribonuclease T1 combines functional and structural features, involving both imidazole nitrogens. To evaluate the use of Asn and Gln, substitutions in dissecting the properties of histidines, we analysed the, consequences of the His92Gln and His92Asn substitutions on the enzyme's, structure, function, and conformational stability by protein engineering, and X-ray crystallographic methods. In the X-ray structures of wild-type, and His92Gln RNase T1 in complex with 2'-GMP the His92-N epsilon 2 and, Gln92-N epsilon 2 atoms are isosterically equivalent. Similarly, the, His92N delta 1H...OAsn99 hydrogen bond observed in wild-type is replaced, by an equivalent Asn92N delta 2H...OAsn99 in the His92Asn mutant, structure. Double mutant cycles at a single position were used to analyse, the intermolecular and intramolecular interactions of the exchangeable, proton and the individual histidine nitrogens. Urea denaturation, measurements as a function of pH revealed that the exchangeable proton of, His92, rather than its imidazole ring is contributing about 1 kcal/mol to, the conformational stability of RNase T1. The stabilizing and the, destabilizing effects of the (His-->Gln) and the (His-->Asn) mutations on, urea denaturation of RNase T1 at pH 9.0 suggest that the unprotonated N, delta 1 and N epsilon 2 atoms contribute in a compensating way to the, conformational stability of RNase T1. A comparative study of the kinetics, of all mutants suggests that the protonated His92 imidazole is a strictly, co-operative catalytic device.

About this Structure

5BIR is a Single protein structure of sequence from Aspergillus oryzae with CA and 2GP as ligands. Active as Ribonuclease T(1), with EC number 3.1.27.3 Structure known Active Site: CAT. Full crystallographic information is available from OCA.

Reference

Dissecting histidine interactions of ribonuclease T1 with asparagine and glutamine replacements: analysis of double mutant cycles at one position., De Vos S, Doumen J, Langhorst U, Steyaert J, J Mol Biol. 1998 Jan 30;275(4):651-61. PMID:9466938

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