Half-of-sites绑定orotidine 5 '磷酸和alpha-D-5-phosphorylribose 1-diphosphate, orotate phosphoribosyltransferase Theorell-Chance酿酒酵母支持小说变体的催化机制与交流网站。

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McClard RW, Holets EA,麦金农,Witte摩根富林明

Half-of-sites绑定orotidine 5 '磷酸和alpha-D-5-phosphorylribose 1-diphosphate, orotate phosphoribosyltransferase Theorell-Chance酿酒酵母支持小说变体的催化机制与交流网站。

生物化学。2006年4月25日,45 (16):5330 - 42。doi: 10.1021 / bi051650o。

PubMed ID

16618122 (在PubMed

]

文摘

乒乓球bi-bi动力学机制归因于酵母orotate phosphoribosyltransferase (OPRTase)[维克多,J。格林伯格,l . B。斯隆,d . l .(1979)生物。化学。254年,2647 - 2655年)已被证明是不起作用(威特,j·F。祖文萃,R。McClard, r . w .(1999)拱门。物化学。Biophys。361年,106 - 112]。放射性标记的orotidine 5 '磷酸(OMP),原位生成从[7 - (14)C] -orotate和alpha-d-5-phoshorylribose 1-diphosphate (PRPP),结合紧密足以OPRTase(由相同的亚基组成的二聚体),复杂的凝胶过滤色谱法。当经济新闻的一个示例。OPRTase广泛透析,1:1(每OPRTase二聚体)(31)P NMR检测到复杂。滴定的酶蛋白OMP收益率(31)P NMR谱峰对自由和enzyme-bound OMP当OMP超过; the complex maintains an OMP/enzyme ratio of 1:1 even when OMP is in substantial excess. A red shift in the UV spectrum of the OMP.OPRTase complex was exploited to measure K(d(OMP)) = 0.84 muM and to verify the 1:1 binding stoichiometry. PRPP forms a Mg(2+)-dependent 1:1 complex with the enzyme as observed by (31)P NMR. Isothermal titration calorimetry (ITC) experiments revealed 1:1 stoichiometries for both OMP and Mg(2+)-PRPP with OPRTase yielding K(d) values of 0.68 and 10 microM, respectively. The binding of either 1 equiv of OMP or PRPP is mutually exclusive. ITC experiments demonstrate that the binding of OMP is largely driven by increased entropy, suggesting substantial distal disordering of the protein. Analytical gel-filtration chromatography confirms that the OMP.OPRTase complex involves the dimeric form of enzyme. The off rate for release of OMP, determined by magnetization inversion transfer, was determined to be 27 s(-)(1). This off rate is somewhat less than the k(cat) in the biosynthetic direction (about 39 s(-)(1)); thus, the release of OMP from OMP.OPRTase may not be kinetically relevant to the steady-state reaction cycle. The body of available data can be explained in terms of alternating site catalysis with either a classical Theorell-Chance mechanism or, far more likely, a novel "double Theorell-Chance" mechanism unique to alternating site catalysis, leading us to propose co-temporal binding of orotate and the release of diphosphate as well as the binding of PRPP and the release of OMP that occur via ternary complexes in alternating site fashion across the two highly cooperative subunits of the enzyme. This novel "double Theorell-Chance" mechanism yields a steady-state rate equation indistinguishable in form from the observed classical ping-pong bi-bi kinetics.

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药物靶点

药物	目标	类	生物	药理作用	行动
5-O-phosphono-alpha-D-ribofuranosyl二磷酸	Orotate phosphoribosyltransferase	蛋白质	鼠伤寒沙门氏菌(应变LT2 / SGSC1412 /写明ATCC 700720)	未知的	底物	细节