Enzyme Kinetic Assay to Measure the Activity of Tumor M2 Pyruvate Kinase in Breast Cancer Patients.

Background: M2 type Pyruvate kinase (PKM2) is the key rate-limiting enzyme of glycolysis, and it mainly exists as a dimer in tumor cells. This study aims to establish an enzyme kinetic assay for serum tumor M2 pyruvate kinase (TuM2-PK), and to evaluate its diagnostic value in breast cancer.

Methods: The catalytic kinetics of Pyruvate Kinase (PK) was examined. Its allosteric regulation property and Michaelis constant were then measured. Next, the levels of TuM2-PK in serum were detected and compared with results from an enzyme-linked immunosorbent assay (ELISA). Finally, the levels of TuM2-PK among breast cancer patients, post-mastectomy patients, patients with benign breast diseases, and healthy controls were compared.

Results: A PK kinetic assay was established in this study. The assay reaction time is 108 seconds, and the optimum pH level is 8.0. In the presence of the allosteric activator fructose 1, 6-bisphosphate (FBP), the Km of PK for phosphoenolpyruvate (PEP) is 0.15 mmol/L and the Vmax is 330 μmol/min. The levels of TuM2-PK in serum obtained by enzyme kinetics are comparable to the ELISA results. Both assays showed that TuM2-PK in breast cancer patients was increased from stage I to IV. Importantly, TuM2-PK levels were significantly different between early and late-stage breast cancer patients (stage I and stage II vs. stage III and IV), as well as between late-stage and non-malignant patients (<0.05). No statistical difference was found between benign breast disease patients and healthy controls.

Conclusions: An enzyme kinetic assay of serum TuM2-PK was successfully established and may be useful for breast cancer diagnosis.