1ay0

From Proteopedia

proteopedia linkproteopedia link

spinqualitylabelsall models 1ay0, resolution 2.6Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image

IDENTIFICATION OF CATALYTICALLY IMPORTANT RESIDUES IN YEAST TRANSKETOLASE

Overview

The possible roles of four histidine residues in the active site of yeast, transketolase were examined by site-directed mutagenesis. Replacement of, the invariant His69 with alanine yielded a mutant enzyme with 1.5% of the, specific activity of the wild-type enzyme and with an increased KM for the, donor. This residue is located at the bottom of the substrate cleft close, to the C1 hydroxyl group of the donor substrate, and the side chain of, His69 might be required for recognition of this hydroxyl group and, possibly for maintenance of the proper orientation of the reaction, intermediate, (alpha, beta-dihydroxyethyl)thiamin diphosphate. Amino acid, replacements of His481 by alanine, serine, and glutamine resulted in, mutant enzymes with significantly increased KM values for the donor, substrate and specific activities of 4.4%, 1.9%, and 5.5% of the wild-type, enzyme. The kinetic data suggest that this residue, although close to the, C2 carbonyl oxygen of the substrate, is not absolutely required for, stabilization of the negative charge that develops at this oxygen in the, transition state. This points toward the 4'-NH2 group of the pyrimidine, ring of thiamin diphosphate as the major source of charge stabilization., Mutations at positions His30 and His263 result in mutant enzymes severely, impaired in catalytic activity (1.5% and less of the activity of wild-type, transketolase). The KM value for the donor substrate was increased for the, His30Ala mutant but remained unchanged in the His263Ala enzyme. The side, chains of both residues interact with the C3 hydroxyl group of the donor, substrate, and the results indicate that the two residues act in concert, during proton abstraction of the C3 hydroxyl proton during catalysis.

About this Structure

1AY0 is a Single protein structure of sequence from Saccharomyces cerevisiae with CA and TPP as ligands. Active as Transketolase, with EC number 2.2.1.1 Known structural/functional Site: . Full crystallographic information is available from OCA.

Reference

Identification of catalytically important residues in yeast transketolase., Wikner C, Nilsson U, Meshalkina L, Udekwu C, Lindqvist Y, Schneider G, Biochemistry. 1997 Dec 16;36(50):15643-9. PMID:9398292

Page seeded by OCA on Tue Dec 18 14:23:16 2007