Bailey Susan, Rapson Trevor, Johnson-Winters Kayunta, Astashkin Andrei V, Enemark John H, Kappler Ulrike
Molecular Biophysics Group, Science and Technology Facilities Council Daresbury Laboratory, Warrington WA4 4AD, United Kingdom.
J Biol Chem. 2009 Jan 23;284(4):2053-63. doi: 10.1074/jbc.M807718200. Epub 2008 Nov 12.
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 Km(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 SDH R55M and SDHH57A are relatively insensitive to pH (approximately 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.
亚硫酸盐脱氢酶（SDHs）催化亚硫酸盐氧化并解毒生成硫酸盐，这一反应对所有生命形式都至关重要。亚硫酸盐氧化酶含有三个保守的活性位点氨基酸（精氨酸-55、组氨酸-57和酪氨酸-236），它们对催化活性至关重要。在此，我们研究了这些残基中两个新的和一个先前报道的取代（R55M、H57A、Y236F）对SDH催化的动力学和结构影响。发现精氨酸-55和组氨酸-57在底物结合中起关键作用。R55M取代使Km（亚硫酸盐）（表观）增加2 - 3个数量级，而当咪唑环完全质子化时，组氨酸-57是在低pH下维持高底物亲和力所必需的。这种效应可能是由组氨酸-57与精氨酸-55的相互作用介导的，这种相互作用稳定了精氨酸-55侧链的位置，或者，也可能反映了亚硫酸盐质子化状态的变化。与SDHWT和SDHY236F不同，SDH R55M和SDHH57A的催化周转率对pH相对不敏感（分别约为60和200 s(-1)）。在结构水平上，显著的动力学效应似乎与无序（在SDHH57A和SDHY236F中）或精氨酸-55的缺失（SDHR55M）相关，这表明精氨酸-55及其参与的氢键相互作用对底物结合和催化至关重要。SDHR55M的结构在活性位点结合有硫酸盐，这一事实与SDHR55M中硫酸盐抑制作用的显著增加相吻合。因此，精氨酸-55似乎也参与了SDH中底物和产物的区分。
