A. Watanabe, T. Yoshimura, B. Mikami
May 24, 2002
Citations
5
Influential Citations
103
Citations
Quality indicators
Journal
The Journal of Biological Chemistry
Abstract
The crystal structures of alanine racemase bound with reaction intermediate analogs,N-(5′-phosphopyridoxyl)-l-alanine (PLP-l-Ala) andN-(5′-phosphopyridoxyl)-d-alanine (PLP-d-Ala), were determined at 2.0-Å 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 Cα among the three structures were less than 0.28 Å. 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 α-hydrogen of the alanine moiety of PLP-l-Ala was located near the OH of Tyr265′, whereas that of PLP-d-Ala was near the NZ of Lys39. These support the previous findings that Tyr265′ and Lys39 are the catalytic bases removing α-hydrogen from l- and d-alanine, respectively. The prerequisite for this two-base mechanism is that the α-proton abstracted from the substrate is transferred (directly or indirectly) between the NZ of Lys39 and the OH of Tyr265′; 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, Lys39 and Tyr265′. The results of molecular orbital calculation also support this mechanism.