Lolkema J S, ten Hoeve-Duurkens R H, Robillard G T
Department of Biochemistry, University of Groningen, The Netherlands.
J Biol Chem. 1993 Aug 25;268(24):17844-9.
The kinetics of mannitol phosphorylation catalyzed by enzyme IImtl of the bacterial P-enolpyruvate-dependent phosphotransferase system are described for three different physical conditions of the enzyme, (i) embedded in the membrane of inside-out (ISO) oriented vesicles, (ii) solubilized and assayed above the critical micellular concentration (cmc) of the detergent, and (iii) solubilized and assayed below the cmc of the detergent. The kinetic characteristics of enzyme IImtl, after solubilization of cytoplasmic membranes or after purification from these membranes are comparable. The mannitol-dependent kinetics at saturating concentration of P-HPr were biphasic both for the solubilized enzyme assayed above the cmc and for the enzyme in ISO vesicles. In contrast, the mannitol-dependent kinetics was monophasic for the solubilized enzyme assayed below the cmc. In the latter case, the maximal rate was about twice as high as observed with the two other conditions. The contribution of the high affinity phase to the maximal rate is lower for enzyme IImtl in ISO vesicles than for the solubilized enzyme. At limiting concentrations of P-HPr, the kinetics is not according to the expected "ping-pong" mechanism.
针对细菌磷酸烯醇式丙酮酸依赖性磷酸转移酶系统的IImtl酶催化甘露醇磷酸化的动力学，描述了该酶在三种不同物理条件下的情况：(i) 嵌入内向外（ISO）取向囊泡的膜中；(ii) 在去污剂的临界胶束浓度（cmc）以上溶解并进行测定；(iii) 在去污剂的cmc以下溶解并进行测定。细胞质膜溶解后或从这些膜中纯化后，IImtl酶的动力学特征具有可比性。在cmc以上测定的溶解酶和ISO囊泡中的酶，在P-HPr饱和浓度下，甘露醇依赖性动力学都是双相的。相比之下，在cmc以下测定的溶解酶，其甘露醇依赖性动力学是单相的。在后一种情况下，最大反应速率约为其他两种条件下观察到的两倍。对于ISO囊泡中的IImtl酶，高亲和力阶段对最大反应速率的贡献低于溶解酶。在P-HPr的极限浓度下，动力学不符合预期的“乒乓”机制。
