Glucose-6-phosphate dehydrogenase from brewers' yeast. The effects of pH and temperature on the steady-state kinetic parameters of the two-chain protein species.
Glucose-6-phosphate dehydrogenase from brewers' yeast. The effects of pH and temperature on the steady-state kinetic parameters of the two-chain protein species.
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来自啤酒酵母的葡萄糖-6-磷酸脱氢酶。
DOI：
10.1021/bi00654a027
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发表时间：
1976
期刊：
影响因子：
2.9
通讯作者：
R. Roy
中科院分区：
文献类型：
--
作者：
S. Kuby;R. Roy
一个系统的研究已经取得了稳定的两个亚基的葡萄糖-6-磷酸脱氢酶的酶物种的稳态动力学参数的pH值和温度依赖性，在叠加的缔合-解离反应的情况下。在pH 5和10之间以及在18-32 ℃下的几种缓冲液中获得的Vmax（app）数据导致以下假设：至少两组质子平衡可以控制催化（一组在25 ℃下接近pH 5.7，另一组在pH 9.2附近）;此外，产物形成的两种途径（即，两个Vmax）似乎需要解释对数Vmax（app）对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，无论是均匀程度的相互作用还是最小程度的缓冲液物质相互作用，都可以从与电离常数的衍生值相关的焓变中进行评估，这些电离常数归因于Tris-乙酸盐-EDTA缓冲液在恒定离子强度下的酶-底物三元复合物中的质子平衡。通过在0.1（T/2）和25和32 ℃下选择该缓冲液，已经出现了自洽的动力学机制，其允许两种完全离子化的底物通过两种主要途径与酶随机结合，并且通过E-A-B-和HE-A-B-形成产物。如前所述（Kuby等人，Arch. Biochem，Biophys. 165，153-178，1974），假定准平衡，在三元络合物的相互转化处具有限速步骤（k + 5和k +5 ′）。由该机制定义的两组质子平衡的值（即，第一次电离的pKk、pKH 2和第二次电离的pKk '、pKH'）。从它们的数值（例如，在25 ℃：pKK = 5.7 PKH 2 = 5.2;和pKK ′ = 9.1，PKH ′ = 8.2）和Δ H度的值（例如，Δ H度pKK约为5.1 KCAL/MOL; Δ H度pKK'约为11 KCAL/MOL），提出了一个推论，将这些酸解离常数分别归因于咪唑和ε-氨基。
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.