Catalytic cofactors (Mg2+ and Zn2+ ions) influence the pattern of vanadate Inhibition of the monoesterase activity of calf intestinal alkaline phosphatase

Abstract

The mechanism of modulation of vanadate inhibition of alkaline phosphatase activity by catalytic cofactors has not been fully characterized. We investigated the effect of the interaction of catalytic cofactors (Mg²⁺ and Zn²⁺) and vanadate (an active site inhibitor) on the rate of hydrolysis of para-nitrophenyl phosphate (pNPP) (monoesterase reaction) by calf intestinal alkaline phosphatase (CIAP). The results showed that vanadate significantly inhibited ʻcofactor-freeʼ CIAP, and the inhibition was relieved by the presence of the catalytic cofactors in the reaction. Our results show that the absence of the cofactors did not significantly alter the K_m of the reaction, but caused a decrease in the V_max. Kinetic analyses showed that vanadate inhibited CIAP-catalyzed hydrolysis of pNPP by decreasing the V_max and increasing the K_m of the reaction. The presence of cofactors in the reaction alleviated the effect of vanadate by increasing the V_max and decreasing the K_m. The activity of the dialyzed CIAP was increased by the addition of catalytic cofactors to vanadate-inhibited enzyme. This study provides preliminary data that reversible inhibition of CIAP is subject to the influence of catalytic cofactors. Further studies will reveal detailed mechanistic aspects of this observation and its significance in the biological system.

Keywords: alkaline phosphatase, monoesterase reaction, vanadate inhibition, catalytic cofactors