Pyruvate kinase from Geobacillus stearothermophilus displays an unusual preference for Mn2+ in a cycling reaction

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 Mn2+ rather than Mg2+. 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 Mn2+ over Mg2+ in the cycling reaction with ATP and GDP or ATP and IDP. The much lower K-m values for the substrate in the presence of Mn2+ rather than Mg2+ are consistent with the results of the cycling reaction observed in this study.