嗜热脂肪地芽孢杆菌的丙酮酸激酶在循环反应中对 Mn 表现出异常的偏好。

Pyruvate kinase from Geobacillus stearothermophilus displays an unusual preference for Mn in a cycling reaction.

机构信息

R&D Group, Diagnostics Dept., Asahi Kasei Pharma Corporation, 632-1 Mifuku, Izunokuni-shi, Shizuoka, 410-2321, Japan.

出版信息

Anal Biochem. 2019 Apr 1;570:27-31. doi: 10.1016/j.ab.2019.02.005. Epub 2019 Feb 7.

DOI:10.1016/j.ab.2019.02.005

Abstract

Previously, we developed a kinase cycling method using creatine kinase and pyruvate kinase (RMPK) both from rabbit muscle in the presence of an excess amount of ATP and IDP for the quantitative determination of substrate. To our surprise, the RMPK cycling reaction was 10-fold more efficient using Mn rather than Mg. Here, we investigated PK from Geobacillus stearothermophilus (GSPK) as an alternative source of enzyme. Spectrophotometric real-time detection was accomplished by coupling the reaction to ADP-dependent glucokinase (ADP-GK) together with glucose-6-phosphate dehydrogenase (G6PD). The rate of increase in absorbance of NADH at 340 nm was monitored. GSPK displayed an even greater preference than RMPK for Mn over Mg in the cycling reaction with ATP and GDP or ATP and IDP. The much lower K values for the substrate in the presence of Mn rather than Mg are consistent with the results of the cycling reaction observed in this study.

摘要

此前，我们开发了一种使用肌酸激酶和丙酮酸激酶（RMPK）的激酶循环方法，该方法来自兔肌肉，在过量 ATP 和 IDP 的存在下用于定量测定底物。令我们惊讶的是，使用 Mn 而不是 Mg 可使 RMPK 循环反应的效率提高 10 倍。在这里，我们研究了来自嗜热脂肪地芽孢杆菌（GSPK）的 PK 作为酶的替代来源。通过将反应与 ADP 依赖性葡萄糖激酶（ADP-GK）和葡萄糖-6-磷酸脱氢酶（G6PD）偶联来实现分光光度实时检测。监测 NADH 在 340nm 处吸光度的增加率。GSPK 在 ATP 和 GDP 或 ATP 和 IDP 的循环反应中对 Mn 的偏好甚至大于 RMPK 对 Mg 的偏好。在 Mn 存在而非 Mg 存在的情况下，底物的 K 值要低得多，这与本研究中观察到的循环反应结果一致。