Mercaptopyruvate Inhibits Tissue-Nonspecific Alkaline Phosphatase and Calcium Pyrophosphate Dihydrate Crystal Dissolution

Abstract

Objective.The enzymatic activities of tissue-nonspecific alkaline phosphatase (TNAP) including capacity to inhibit calcium pyrophosphate dihydrate (CPPD) crystal dissolution are known to be inhibited by endogenous amino acids, notably cysteine. As cysteine is recognized as a strong TNAP inhibitor, we investigated whether cysteine-related metabolites such as mercaptopyruvate (MPA) could show similar enzyme inhibition effects and, if so, whether these effects might be synergistic with cysteine at approximate physiologic concentrations of the amino acids.Methods.We studied the inhibitory effects of MPA as well as MPA and cysteine combined in equimolar concentrations on TNAP’s phosphatase, inorganic pyrophosphatase, and CPPD crystal dissolution activities. Kinetic parameters Vmax, KM, concentration for 50% inhibition (I50), inhibitor constant (KI), and specific activities calculated from initial velocity, Eadie-Hofstee, Simple, Dixon, and secondary plots were used to assess enzyme inhibition.Results.MPA significantly inhibited TNAP’s phosphatase and pyrophosphatase activities at 10× and 100× physiological concentrations. In the presence of calcium [Ca2+] and [Mg2+] = 1 mM, MPA inhibited uncompetitively TNAP’s phosphatase activity and inhibited noncompetitively its pyrophosphatase activity. CPPD crystal dissolution activity was also inhibited. Cysteine and MPA together in equimolar concentrations inhibited TNAP enzyme activities and CPPD crystal dissolution much more effectively than MPA or cysteine alone, reducing CPPD dissolution to 38% of controls at approximate physiologic inhibitor concentrations.Conclusion.Endogenous amino acids like cysteine and its derivative MPA have the capacity to inhibit TNAP activities at physiologic concentrations. Downregulation of their inhibiting concentration in the cartilage interstitial fluid environment may provide a therapeutic avenue to controlled dissolution of CPPD crystal deposition in tissues.