spacer Alanine racemase bound with N-(5'-phosphopyridoxyl)-D-alanine
Primary citation
Title Reaction mechanism of alanine racemase from Bacillus stearothermophilus: x-ray crystallographic studies of the enzyme bound with N-(5'-phosphopyridoxyl)alanine.
Authors Watanabe,; Yoshimura,; Mikami,; Hayashi,; Kagamiyama,; Esaki,
Journal vol:277, pag:19166-19172 (2002), Identifiers: PubMed ID (11886871)search DOI (10.1074/jbc.M201615200)
Abstract The crystal structures of alanine racemase bound with reaction intermediate analogs, N-(5'-phosphopyridoxyl)-L-alanine (PLP-L-Ala) and N-(5'-phosphopyridoxyl)-D-alanine (PLP-D-Ala), were determined at 2.0-A resolution with the crystallographic R factor of 17.2 for PLP-L-Ala and 16.9 for PLP-D-Ala complexes. They were quite similar not only to each other but also to the structure of the native pyridoxal 5'-phosphate (PLP)-form enzyme; root mean square deviations at Calpha among the three structures were less than 0.28 A. The side chains of the amino acid residues around the PLP-L-Ala and PLP-D-Ala were virtually superimposable on each other as well as on those around PLP of the native holoenzyme. The alpha-hydrogen of the alanine moiety of PLP-L-Ala was located near the OH of Tyr(265)', whereas that of PLP-D-Ala was near the NZ of Lys(39). These support the previous findings that Tyr(265)' and Lys(39) are the catalytic bases removing alpha-hydrogen from L- and D-alanine, respectively. The prerequisite for this two-base mechanism is that the alpha-proton abstracted from the substrate is transferred (directly or indirectly) between the NZ of Lys(39) and the OH of Tyr(265'); otherwise the enzyme reaction stops after a single turnover. Only the carboxylate oxygen atom of either PLP-Ala enantiomer occurred at a reasonable position that can mediate the proton transfer; neither the amino acid side chains nor the water molecules were located in the vicinity. Therefore, we propose a mechanism of alanine racemase reaction in which the substrate carboxyl group directly participates in the catalysis by mediating the proton transfer between the two catalytic bases, Lys(39) and Tyr(265)'. The results of molecular orbital calculation also support this mechanism.
MeSH terms Alaninesearch, Alanine Racemasesearch, Binding Sitessearch, Catalysissearch, Crystallographysearch, X-Raysearch, Geobacillus stearothermophilussearch, Modelssearch, Molecularsearch, Protein Conformationsearch
Other entries described in this publication 1l6f
Secondary citations
Title Determination of the structure of alanine racemase from Bacillus stearothermophilus at 1.9-A resolution
Authors Shaw,; Petsko,; Ringe,
Journal BIOCHEMISTRYsearch vol:36, pag:1329-1342 (1997) DOI (10.1021/bi961856c)
Title Reaction of alanine racemase with 1-aminoethylphosphonic acid forms a stable external aldimine
Authors Stamper,; Morollo,; Ringe,
Journal BIOCHEMISTRYsearch vol:37, pag:10438-10445 (1998) DOI (10.1021/bi980692s)
Title Structure of a Michaelis complex analogue: propionate binds in the substrate carboxylate site of alanine racemase
Authors Morollo,; Petsko,; Ringe,
Journal BIOCHEMISTRYsearch vol:38, pag:3293-3301 (1999) DOI (10.1021/bi9822729)