Molecular Basis for Enzymatic Sulfite Oxidation HOW THREE CONSERVED ACTIVE SITE RESIDUES SHAPE ENZYME ACTIVITY

被引:30
作者
Bailey, Susan [2 ]
Rapson, Trevor [1 ]
Johnson-Winters, Kayunta [3 ]
Astashkin, Andrei V. [3 ]
Enemark, John H. [3 ]
Kappler, Ulrike [1 ]
机构
[1] Univ Queensland, Sch Mol & Microbial Sci, Ctr Met Biol, Brisbane, Qld 4072, Australia
[2] Daresbury Lab, Sci & Technol Facil Council, Mol Biophys Grp, Warrington WA4 4AD, Cheshire, England
[3] Univ Arizona, Dept Chem, Tucson, AZ 85721 USA
基金
英国科学技术设施理事会; 美国国家卫生研究院;
关键词
INTRAMOLECULAR ELECTRON-TRANSFER; CYTOCHROME-C OXIDOREDUCTASE; RHODOBACTER-CAPSULATUS; PULSED EPR; OXIDASE; BIOLOGY; EXPRESSION; CATALYSIS; INSIGHTS; COMPLEX;
D O I
10.1074/jbc.M807718200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Sulfite dehydrogenases (SDHs) catalyze the oxidation and detoxification of sulfite to sulfate, a reaction critical to all forms of life. Sulfite-oxidizing enzymes contain three conserved active site amino acids (Arg-55, His-57, and Tyr-236) that are crucial for catalytic competency. Here we have studied the kinetic and structural effects of two novel and one previously reported substitution (R55M, H57A, Y236F) in these residues on SDH catalysis. Both Arg-55 and His-57 were found to have key roles in substrate binding. An R55M substitution increased K-m(sulfite)(app) by 2-3 orders of magnitude, whereas His-57 was required for maintaining a high substrate affinity at low pH when the imidazole ring is fully protonated. This effect may be mediated by interactions of His-57 with Arg-55 that stabilize the position of the Arg-55 side chain or, alternatively, may reflect changes in the protonation state of sulfite. Unlike what is seen for SDHWT and SDHY236F, the catalytic turnover rates of SDHR55M and SDHH57A are relatively insensitive to pH (similar to 60 and 200 s(-1), respectively). On the structural level, striking kinetic effects appeared to correlate with disorder (in SDHH57A and SDHY236F) or absence of Arg-55 (SDHR55M), suggesting that Arg-55 and the hydrogen bonding interactions it engages in are crucial for substrate binding and catalysis. The structure of SDHR55M has sulfate bound at the active site, a fact that coincides with a significant increase in the inhibitory effect of sulfate in SDHR55M. Thus, Arg-55 also appears to be involved in enabling discrimination between the substrate and product in SDH.
引用
收藏
页码:2053 / 2063
页数:11
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