Hengge A C, Sowa G A, Wu L, Zhang Z Y
Institute for Enzyme Research, University of Wisconsin, Madison 53705, USA.
Biochemistry. 1995 Oct 31;34(43):13982-7. doi: 10.1021/bi00043a003.
The dephosphorylation of p-nitrophenyl phosphate by Yersinia protein-tyrosine phosphatase (PTPase) and by the rat PTP1 has been examined by measurement of heavy-atom isotope effects at the nonbridge oxygen atoms [18(V/K)nonbridge], at the bridging oxygen atom [18(V/K)bridge], and the nitrogen atom in the leaving group 15(V/K). The effects were measured using an isotope ratio mass spectrometer by the competitive method and thus are effects on V/K. The results for the Yersinia PTPase and rat PTP1, respectively, are 1.0142 +/- 0.0004 and 1.0152 +/- 0.0006 for 18(V/K)bridge; 0.9981 +/- 0.0015 and 0.9998 +/- 0.0013 for 18(V/K)nonbridge; and 1.0001 +/- 0.0002 and 0.9999 +/- 0.0003 for 15(V/K). The magnitudes of the isotope effects are similar to the intrinsic values measured in solution, indicating that the chemical step is rate-limiting for V/K. The transition state for phosphorylation of the enzyme is dissociative in character, as is the case in solution. Binding of the substrate is rapid and reversible, as is the binding-induced conformational change which brings the catalytic general acid into the active site. Cleavage of the P-O bond and proton transfer from the general acid Asp to the leaving group are both far advanced in the transition state, and there is no development of negative charge on the departing leaving group. Experiments with several general acid mutants give values for 18(V/K)bridge of around 1.0280, 15(V/K) of about 1.002, and 18(V/K)nonbridge effects of from 1.0007 to 1.0022. These data indicate a dissociative transition state with the leaving group departing as the nitrophenolate anion but suggest more nucleophilic participation than in the solution reaction.
通过测量重原子同位素效应，即在非桥氧原子处的[18(V/K)非桥]、桥氧原子处的[18(V/K)桥]以及离去基团中氮原子处的15(V/K)，对耶尔森氏菌蛋白酪氨酸磷酸酶（PTPase）和大鼠PTP1催化对硝基苯磷酸酯的去磷酸化反应进行了研究。这些效应通过竞争法利用同位素比率质谱仪进行测量，因此是对V/K的效应。耶尔森氏菌PTPase和大鼠PTP1的结果分别为：18(V/K)桥为1.0142±0.0004和1.0152±0.0006；18(V/K)非桥为0.9981±0.0015和0.9998±0.0013；15(V/K)为1.0001±0.0002和0.9999±0.0003。同位素效应的大小与在溶液中测量的本征值相似，表明化学步骤是V/K的限速步骤。酶磷酸化的过渡态具有解离特征，这与溶液中的情况相同。底物的结合迅速且可逆，结合诱导的构象变化也是如此，该变化将催化性的通用酸带入活性位点。在过渡态中，P - O键的断裂和质子从通用酸天冬氨酸转移到离去基团都已进行到相当程度，并且离去的离去基团上没有负电荷的形成。对几种通用酸突变体的实验给出的18(V/K)桥值约为1.0280，15(V/K)约为1.002，18(V/K)非桥效应在1.0007至1.0022之间。这些数据表明存在一个解离过渡态，离去基团以硝基酚酸根阴离子的形式离去，但表明亲核参与比溶液反应中更多。
