1dpb
From Proteopedia
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CRYSTALLOGRAPHIC AND ENZYMATIC INVESTIGATIONS ON THE ROLE OF SER558, HIS610 AND ASN614 IN THE CATALYTIC MECHANISM OF AZOTOBACTER VINELANDII DIHYDROLIPOAMIDE ACETYLTRANSFERASE (E2P)
Overview
Dihydrolipoamide acetyltransferase (E2p) is the structural and catalytic, core of the pyruvate dehydrogenase multienzyme complex. In Azotobacter, vinelandii E2p, residues Ser558, His610', and Asn614' are potentially, involved in transition state stabilization, proton transfer, and, activation of proton transfer, respectively. Three active site mutants, S558A, H610C, and N614D, of the catalytic domain of A. vinelandii E2p were, prepared by site-directed mutagenesis and enzymatically characterized. The, crystal structures of the three mutants have been determined at 2.7, 2.5, and 2.6 A resolution, respectively. The S558A and H610C mutants exhibit a, strongly (200-fold and 500-fold, respectively) reduced enzymatic activity, whereas the substitution of Asn614' by aspartate results in a moderate, (9-fold) reduced activity. The decrease in enzymatic activity of the S558A, and H610C mutants is solely due to the absence of the hydroxyl and, imidazole side chains, respectively, and not due to major conformational, rearrangements of the protein. Furthermore the sulfhydryl group of Cys610', is reoriented, resulting in a completely buried side chain which is quite, different from the solvent-exposed imidazole group of His610' in the, wild-type enzyme. The presence of Asn614' in A. vinelandii E2p is, exceptional since all other 18 known dihydrolipoamide acyltransferase, sequences contain an aspartate in this position. We observe no difference, in conformation of Asp614' in the N614D mutant structure compared with the, conformation of Asn614' in the wild-type enzyme. Detailed analysis of all, available structures and sequences suggests two classes of, acetyltransferases: one class with a catalytically essential His-Asn pair, and one with a His-Asp-Arg triad as present in chloramphenicol, acetyltransferase [Leslie, A. G. W. (1990) J. Mol. Biol. 213, 167-186] and, in the proposed active site models of Escherichia coli and yeast E2p.
About this Structure
1DPB is a Single protein structure of sequence from Azotobacter vinelandii. Active as Dihydrolipoyllysine-residue acetyltransferase, with EC number 2.3.1.12 Full crystallographic information is available from OCA.
Reference
Crystallographic and enzymatic investigations on the role of Ser558, His610, and Asn614 in the catalytic mechanism of Azotobacter vinelandii dihydrolipoamide acetyltransferase (E2p)., Hendle J, Mattevi A, Westphal AH, Spee J, de Kok A, Teplyakov A, Hol WG, Biochemistry. 1995 Apr 4;34(13):4287-98. PMID:7703242
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