Examination of the transition state of the low-molecular mass small tyrosine phosphatase .1. Comparisons with other protein phosphatases
Examination of the transition state of the low-molecular mass small tyrosine phosphatase .1. Comparisons with other protein phosphatases
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DOI：
10.1021/bi970364c
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发表时间：
1997-06-24
期刊：
影响因子：
2.9
通讯作者：
Zhang, ZY
中科院分区：
文献类型：
--
作者：
Hengge, AC;Zhao, Y;Zhang, ZY
用重原子同位素效应研究了磷酸对硝基苯酯（pNPP）与低分子量酪氨酸磷酸酶Stp 1及其突变体D128 N、D128 A、D128 E和S18 A的反应。在非桥氧原子[（18）（V/K）（非桥）]、桥氧原子（键断裂位点）[（18）（V/K）（桥）]和硝基苯酚离去基团中的氮原子[（15）（V/K）]处测量同位素效应。天然Stp 1的结果为1.0160 +/- 0.0005（18）（V/K）（桥接）、1.0007 +/- 0.0001（15）（V/K）和1.0018 +/- 0.0003（18）（V/K）（非桥接）。（18）（V/K）（非桥）和（15）（V/K）的值不同于先前用其它蛋白酪氨酸磷酸酶测量的值，也不同于pNPP的水水解反应的值。这些值表明，在过渡态的天然Stp 1反应的离去基团带有部分负电荷，并有亲核相互作用之间的半胱氨酸亲核试剂，和磷酰基，导致一些非桥P-O键顺序的减少。过渡态相对于键断裂成离去基团的程度保持高度解离。一般的酸从天冬氨酸突变为谷氨酸会减慢催化作用，但不会引起同位素效应的变化，因此不会改变质子转移到过渡态离去基团的程度。该残基突变为天冬酰胺或丙氨酸，18（V/K）（桥）的值约为1.029，（15）（V/K）的值约为1.003，（18）（V/K）的值约为1.003。（非桥）1.0010（D128 A）至1.0024（D128 N），这些数据表明了一个解离的过渡态，离去基团作为硝基酚盐阴离子离开，并表明比在水溶液中更多的亲核参与与S18 A突变体的同位素效应，其中阴离子Cys亲核试剂的氢键稳定性已被删除，与天然酶的实验误差范围内，表明这种改变对从PNPP的磷酰基转移的过渡态没有影响。
The reactions of p-nitrophenyl phosphate (pNPP) with the low-molecular mass tyrosine phosphatase Stp1 and with the mutants D128N, D128A, D128E, and S18A have been studied by measurement of heavy-atom isotope effects in the substrate. The isotope effects were measured at the nonbridging oxygen atoms [(18)(V/K)(nonbridge)], at the bridging oxygen atom (the site of bond cleavage) [(18)(V/K)(bridge)], and at the nitrogen atom in the nitrophenol leaving group [(15)(V/K)]. The results with native Stp1 were 1.0160 +/- 0.0005 for (18)(V/K)(bridge), 1.0007 +/- 0.0001 for (15)(V/K), and 1.0018 +/- 0.0003 for (18)(V/K)(nonbridge). The values for (18)(V/K)(nonbridge) and (15)(V/K) differ from those previously measured with other protein-tyrosine phosphatases and from those of the aqueous hydrolysis reaction of pNPP. The values indicate that in the transition state of the native Stp1 reaction the leaving group bears a partial negative charge, and there is nucleophilic interaction between the Cys nucleophile, and the phosphoryl group, causing some decrease in the nonbridge P-O bond order. The transition state remains highly dissociative with respect to the degree of bond cleavage to the leaving group. Mutation of the general acid from aspartic acid to glutamic acid slows catalysis but causes no change in the isotope effects and thus does not alter the degree of proton transfer to the leaving group in the transition state. Mutations of this residue to asparagine or alanine give values for 18(V/K)(bridge) Of about 1.029, for (15)(V/K) of about 1.003, and for (18)(V/K)(nonbridge) Of 1.0010 (D128A) to 1.0024 (D128N), These data indicate a dissociative transition state with the leaving group departing as the nitrophenolate anion and indicate more nucleophilic participation than in the aqueous hydrolysis of the pNPP dianion, just as in the native enzyme, The isotope effects with the S18A mutant, in which a hydrogen bonding stabilization of the anionic Cys nucleophile has been removed, were within experimental error of those with the native enzyme, indicating that this alteration has no effect on the transition state for phosphoryl transfer from PNPP.