Background: Enzyme cycling is a sensitive assay method that exploits amplification techniques. We recently
developed a novel enzymatic cycling method, which utilizes both the forward and reverse reactions
of creatine kinase (CK), for the quantitative determination of creatine.
Objective: Our aim was to develop a novel enzymatic cycling method to measure pyruvate or phosphoenolpyruvate
using pyruvate kinase (PK), which exploits a similar principle to that of the CK assay.
Methods: The method was developed by utilizing the reversibility of the PK reaction, in which the forward
(pyruvate forming) and reverse reactions are catalyzed in the presence of an excess amount of IDP
and ATP. Spectrophotometric real-time detection was accomplished by coupling the reaction to ADPdependent
glucokinase (ADP-GK) together with glucose-6-phosphate dehydrogenase (G6PD). The rate
of increase in absorbance of NADH at 340 nm was monitored.
Results: When using PK from rabbit muscle (RMPK), we observed that the efficiency of the cycling reaction
was markedly enhanced when Mg2+ was replaced with Mn2+. Unexpectedly, steady-state kinetic
analysis of the reverse reaction alone coupled with ADP-GK and G6PD revealed the initial velocity was
accelerated by approximately three-fold upon addition of IDP or GDP. This activation was not observed
when Mg2+ was used as the divalent cation.
Conclusion: We have developed a novel enzymatic cycling method mediated by RMPK, which utilizes
the reversibility of the reaction in the presence of an excess amount of ATP and IDP with Mn2+. The
limit of detection was estimated to be 12 nM.