Kuby S A, Roy R N
Biochemistry. 1976 May 4;15(9):1975-87. doi: 10.1021/bi00654a027.
A systematic study has been made of the pH- and temperature-dependency of the steady-state kinetic parameters of the stabilized two-subunit enzyme species of glucose-6-phosphate dehydrogenase, in the absence of superimposed association-dissociation reactions. The Vmax(app) data obtained in several buffers between pH 5 and 10 and at 18-32 degrees C lead to the postulate that at least two sets of protonic equilibria may govern the catalysis (one near pH 5.7 AT 25 DEGREES C and another near pH 9.2); furthermore, two pathways for product formation (i.e., two Vmax's) appear to be required to explain the biphasic nature of the log Vmax(app) vs. pH curves, with Vmax(basic) greater than Vmax(acidic + neutral). Of the several buffers explored, either a uniform degree of interaction or a minimal degree of buffer species interaction could be assessed from the enthalpy changes associated with the derived values for ionization constants attributed to the protonic equilibria in the enzyme-substrates ternary complexes for the case of Tris-acetate-EDTA buffers, at constant ionic strength. With the selection of this buffer at 0.1 (T/2) and at 25 and 32 degrees C, a self-consistent kinetic mechanism has emerged which allows for the random binding of the two fully ionized substrates to the enzyme via two major pathways, and product formation by both E-A--B- and HE-A--B-. As before (Kuby et al. Arch. Biochem, Biophys. 165, 153-178, 1974), a quasi-equilibrium is presumed, with rate-limiting steps (k + 5 and k + 5') at the interconversion of the ternary complexes. Values for the two sets of protonic equilibria defined by this mechanism (viz., pKk, pKH2 for the first ionizations, and pKk', pKH' for the second) could then be estimated. From their numerical values (e.g., at 25 degrees C: pKK = 5.7 PKH2 = 5.2; and pKK' = 9.1, PKH' = 8.2) and from the values for delta H degrees ioniz (e.g., delta H degrees pKK APPROXIMATELY 5.1 KCAL/MOL; DELTA H degrees pKK' APPROXIMATELY 11 KCAL/MOL), A POSTULATE IS PRESENTED WHICH ATTRIBUTES THESE Acid dissociation constants to an imidazole and epsilon-amino group, respectively.
在不存在叠加的缔合-解离反应的情况下，对葡萄糖-6-磷酸脱氢酶稳定的二亚基酶物种的稳态动力学参数的pH和温度依赖性进行了系统研究。在pH 5至10的几种缓冲液中以及在18-32℃下获得的Vmax（app）数据表明，至少两组质子平衡可能控制催化作用（一组在25℃时接近pH 5.7，另一组接近pH 9.2）；此外，似乎需要两条产物形成途径（即两个Vmax）来解释log Vmax（app）与pH曲线的双相性质，其中Vmax（碱性）大于Vmax（酸性+中性）。在探索的几种缓冲液中，对于Tris-乙酸-EDTA缓冲液的情况，在恒定离子强度下，可以从与归因于酶-底物三元复合物中质子平衡的电离常数的推导值相关的焓变中评估均匀程度的相互作用或最小程度的缓冲物种相互作用。选择该缓冲液在0.1（T/2）以及25和32℃下，出现了一种自洽的动力学机制，该机制允许两种完全电离的底物通过两条主要途径随机结合到酶上，并通过E-A--B-和HE-A--B-形成产物。如前所述（Kuby等人，《生物化学与生物物理学档案》，165，153-178，1974），假定为准平衡，在三元复合物的相互转化中有速率限制步骤（k + 5和k + 5'）。然后可以估计由该机制定义的两组质子平衡的值（即，第一次电离的pKk、pKH2，以及第二次电离的pKk'、pKH'）。从它们的数值（例如，在25℃时：pKK = 5.7，PKH2 = 5.2；pKK' = 9.1，PKH' = 8.2）以及δH°电离的值（例如，δH°pKK约为5.1千卡/摩尔；δH°pKK'约为11千卡/摩尔），提出了一种假设，将这些酸解离常数分别归因于一个咪唑基和一个ε-氨基。
