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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 ‘cofactor-free’ 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 Km of the reaction, but caused a decrease in the Vmax. Kinetic analyses showed that vanadate inhibited CIAP-catalyzed hydrolysis of pNPP by decreasing the Vmax and increasing the Km of the reaction. The presence of cofactors in the reaction alleviated the effect of vanadate by increasing the Vmax and decreasing the Km. 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.

The inactivation of peroxidases by its oxidant substrate H2O2 limits the usefulness of these versatile enzymes. Here, we investigated the effect of reaction conditions on inactivation of horseradish peroxidase by excess H2O2. Inactivation was more pronounced at pH extremes, indicating that reactions in which the oxidation products induce significant changes in reaction pH could accentuate the loss of peroxidase activity. In reactions carried out in sodium acetate buffer, higher inactivation rates were observed when the buffer ion concentration was increased, an indication that peroxidase might be generating reactive radicals from the buffer molecules. Promethazine exerted a modest protective effect against inactivation; however, higher concentrations of the redox mediator caused a slight increase in inactivation, likely due to the formation of reactive promethazine radicals, which in turn attack the protein via a mechanism different from that caused by excess H2O2. These findings will help in defining the optimal reaction conditions that preserve the activity of the peroxidase molecules.

The interactions of Mg2+ and Zn2+ ions in the activation of non-specific tissue alkaline phosphatase were investigated using crude extracts of rat kidney. Activation of alkaline phosphatase by the metal ions was accompanied by changes in the kinetic parameters of p-nitrophenylphosphate hydrolysis. The results suggest some synergistic interactions between Mg2+ and Zn2+ ionsin promoting the hydrolysis of p-nitrophenylphosphate by alkaline phosphatase. The results show that assays of alkaline phosphatase activity in homogenised tissue samples will give better responses if both Mg2+ and Zn2+ ions are included in the reactions

The concentration-dependent stimulation of rat liver alkaline phosphatase (ALP) catalyzed hydrolysis of para- nitrophenylphosphate (pNPP) was studied. ALP displayed some activity even in the absence of exogenous Mg2+. Kinetic analyses show that activation by Mg2+ is exerted at the Vmax level without necessarily enhancing the affinity of the enzyme for the ion. However, the hyperbolic activation operates only within the optimal level of 0 to 5mM concentrations of the metal ion. Higher concentrations were actually inhibitory in a pure non-competitive manner. Mg2+, either as an activator (optimal concentrations) or inhibitor (supra-optimal levels) exerts its action via a Vmax effect with only negligible effect on Kmfor the substrate.

The combined effect of two linear inhibitors of rat liver alkaline phosphatase (ALP). vanadate (Van) and L-phenylalanine (L-phe) were studied using a modification of the common Yonetani-Theorell procedure proposed for studying synergistic inhibition. The modes of inhibition of ALP by Van and L-phe as analysed using the double reciprocal plots of the Michaelis-Menten equation were mixed and uncompetitive inhibition respectively. Analysis of the combined effect of the two inhibitors showed that their inhibitory effects were mutually enhanced. The mechanistic aspects and practical applications of the procedure are discussed.