Enzymatic control of cofactor chemistry: Mechanisms of hydrogen ion transfer in alanine racemase and xylose isomerase

The requirement for D-alanine in the peptidoglycan layer of bacterial cell walls is fulfilled by alanine racemase (EC 5.1.1.1), a pyridoxal 5'-phosphate (PLP) assisted enzyme. The enzyme utilizes two antiparallel bases focused at the Calpha position and oriented perpendicular to the PLP ring in order to facilitate the equilibration of alanine enantiomers. As it is a bacterial enzyme, understanding how this two base system is utilized and controlled to yield reaction specificity is therefore a potential means for designing antibiotics. Cycloserine presents an ideal test case, as it represents a widely known yet poorly understood suicide substrate of alanine racemase. Through spectroscopic assays, evidence is provided of a pyridoxal derivative (arising from either isomer of cycloserine) saturated at the C4' carbon position. Additionally; the D- and L-cycloserine inactivated crystal structures of Bacillus stearotherinophilus alanine racemase corroborate the spectroscopy through evidence of a 3-hydroxyisoxazole pyridoxamine derivative. Further, the effects of a Y265F mutation are also assessed in terms of structure and kinetics, suggesting roles for the two catalytic bases in cycloserine inactivation. Based upon the kinetic and structural properties of both the D- and L-isomers of the inhibitor, a mechanism of alanine racemase inactivation by cycloserine is proposed. This pathway involves an initial transamination step followed by tautomerization to form a stable aromatic adduct, a scheme similar to that seen in cycloserine inactivation of aminotransferases.;Additional experiments detail the atomic resolution structure of xylose isomerase (EC 5.3.1.5), an enzyme with an absolute requirement for two divalent cations at its active site to drive the hydride transfer portion of sugar isomerization. Evidence suggests some degree of metal movement at the second metal site, although how this movement may affect catalysis is unknown. The 0.86A resolution structure presented here displays three alternative positions for the second metal site, only one of which appears positioned in a catalytically competent manner. The large degree of disorder at the catalytic site suggests a mechanism for inefficient catalysis in comparison to proton transfer mechanisms. Based on these results, a modified version of the bridged bimetallic mechanism for hydride transfer in the case of Streptomyces olivochromogenes xylose isomerase is proposed. (Abstract shortened by UMI.).