1DPB

CRYSTALLOGRAPHIC AND ENZYMATIC INVESTIGATIONS ON THE ROLE OF SER558, HIS610 AND ASN614 IN THE CATALYTIC MECHANISM OF AZOTOBACTER VINELANDII DIHYDROLIPOAMIDE ACETYLTRANSFERASE (E2P)

1DPB の概要

• エントリーDOI: 10.2210/pdb1dpb/pdb
• 分子名称: DIHYDROLIPOYL-TRANSACETYLASE (2 entities in total)
• 機能のキーワード: dihydrolipoamide acetyltransferase
• 由来する生物種: Azotobacter vinelandii
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 26206.68

構造登録者

Hendle, J.,Hol, W.G.J. (登録日: 1995-02-03, 公開日: 1995-04-20, 最終更新日: 2024-02-07)

主引用文献

Hendle, J.,Mattevi, A.,Westphal, A.H.,Spee, J.,de Kok, A.,Teplyakov, A.,Hol, W.G.
Crystallographic and enzymatic investigations on the role of Ser558, His610, and Asn614 in the catalytic mechanism of Azotobacter vinelandii dihydrolipoamide acetyltransferase (E2p).
Biochemistry, 34:4287-4298, 1995

PubMed Abstract: 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.

PubMed: 7703242
DOI: 10.1021/bi00013a018

実験手法

X-RAY DIFFRACTION (2.5 Å)