TY  - JOUR
A1  - Hall, James
A1  - Reschke, Stefan
A1  - Cao, Hongnan
A1  - Leimkühler, Silke
A1  - Hille, Russ
T1  - The reductive half-reaction of xanthine dehydrogenase from rhodobacter capsulatus the role of GLU(232) in catalysis
JF  - The journal of biological chemistry
N2  - Background: Kinetic characterization of wild-type xanthine dehydrogenase and variants. Results: Comparison of the pH dependence of both k(red) and k(red)/K-d, as well as k(cat) and k(cat)/K-m. Conclusion: Ionized Glu(232) of wild-type enzyme plays an important role in catalysis by discriminating against the monoanionic form of xanthine. Significance: Examining the contributions of Glu(232) to catalysis is essential for understanding the mechanism of xanthine dehydrogenase.
 The kinetic properties of an E232Q variant of the xanthine dehydrogenase from Rhodobacter capsulatus have been examined to ascertain whether Glu(232) in wild-type enzyme is protonated or unprotonated in the course of catalysis at neutral pH. We find that k(red), the limiting rate constant for reduction at high [xanthine], is significantly compromised in the variant, a result that is inconsistent with Glu(232) being neutral in the active site of the wild-type enzyme. A comparison of the pH dependence of both k(red) and k(red)/K-d from reductive half-reaction experiments between wild-type and enzyme and the E232Q variant suggests that the ionized Glu(232) of wild-type enzyme plays an important role in catalysis by discriminating against the monoanionic form of substrate, effectively increasing the pK(a) of substrate by two pH units and ensuring that at physiological pH the neutral form of substrate predominates in the Michaelis complex. A kinetic isotope study of the wild-type R. capsulatus enzyme indicates that, as previously determined for the bovine and chicken enzymes, product release is principally rate-limiting in catalysis. The disparity in rate constants for the chemical step of the reaction and product release, however, is not as great in the bacterial enzyme as compared with the vertebrate forms. The results indicate that the bacterial and bovine enzymes catalyze the chemical step of the reaction to the same degree and that the faster turnover observed with the bacterial enzyme is due to a faster rate constant for product release than is seen with the vertebrate enzyme.
KW  - Enzyme Kinetics
KW  - Glutamate
KW  - Glutamine
KW  - Isotope Effect
KW  - Ultraviolet-visible Spectroscopy (UV-visible Spectroscopy)
KW  - Xanthine
KW  - Xanthine Dehydrogenase
KW  - Xanthine Oxidase
KW  - pH Dependence
Y1  - 2014
U6  - https://doi.org/10.1074/jbc.M114.603456
SN  - 0021-9258
SN  - 1083-351X
VL  - 289
IS  - 46
SP  - 32121
EP  - 32130
PB  - American Society for Biochemistry and Molecular Biology
CY  - Bethesda
ER  -