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首页> 外文期刊>Biochemistry >Contribution of Enzyme-Phosphoribosyl Contacts to Catalysis by Orotidine 5'-Phosphate Decarboxylase
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Contribution of Enzyme-Phosphoribosyl Contacts to Catalysis by Orotidine 5'-Phosphate Decarboxylase

机译：酶-磷酸核糖基接触对Orotidine 5'-磷酸脱羧酶催化的贡献

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摘要

The crystal structure of the complex formed between recombinant yeast orotidine 5'-phosphate decarboxylase and the competitive inhibitor 6-hydroxyuridine 5'-phosphate reveals the presence of four hydrogen bonds between active site residues Tyr-217 and Arg-235 and the phosphoryl group of this inhibitor. When Try-217 and Arg-235 are individually mutated to alanine, values of k_cat/K_m are reduced by factors of 3000- and 7300-fold, respectively. In the Y217A/R235A doble mutant, activity is reduced more than 10~7-fold. Experiments with highly enriched [~14C] orotin acid show that when ribose 5'-phosphate is deleted from substrate orotidine 5'-phosphate, k_cat/K_m is reduced by more than 12 orders of magnitude, from 6.3 * 10~7 M~(-1) s~(-1) for OMP to less than 2.5 * 10~(-5) M(-1) s~(-1) for orotic acid. Activity toward orotate is not "rescued" by 1 M inorganic phosphate. The K_i value of ribose 5'-phosphate, representing the part of the natural substrate that is absent in orotic acis, is 8.1 * 10~(-5) M. Thus, the effective concentration of the 5'-phosphoribosyl group, in stabilizing the transition state for enzymatic decarboxylation of OMP, is estimated to be >2 * 10~8 M, representing one of the largest connectivity effects that has been reported for an enzyme reaction.
机译：重组酵母维他命5'-磷酸脱羧酶和竞争性抑制剂6-羟基尿苷5'-磷酸之间形成的复合物的晶体结构表明，在活性位点残基Tyr-217和Arg-235与磷酸化的磷酸基之间存在四个氢键这种抑制剂。当Try-217和Arg-235分别突变为丙氨酸时，k_cat / K_m的值分别降低了3000倍和7300倍。在Y217A / R235A双突变体中，活性降低了10〜7倍以上。高浓缩[〜14C]牛磺酸的实验表明，当从核苷5'-磷酸中删除核糖5'-磷酸时，k_cat / K_m降低了12个数量级，从6.3 * 10〜7 M〜（ OMP的-1）s〜（-1）小于乳清酸的2.5 * 10〜（-5）M（-1）s〜（-1）。 1 M无机磷酸盐不能“挽救”对乳清酸盐的活性。核糖5'-磷酸的K_i值（代表天然底物中不存在部分顺式的部分）的K_i值为8.1 * 10〜（-5）M。因此，稳定化过程中有效的5'-磷酸核糖基浓度OMP的酶促脱羧过渡态估计为> 2 * 10〜8 M，代表了酶反应中最大的连通性效应之一。

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