Abstract
Abstract The degree of inhibition of rat intestinal alkaline phosphatase by l-phenylalanine was highly pH-dependent and varied from 0 to 66% within a pH range of 7.8 to 10.4, exhibiting a peak at pH 9.2 and 8.7 for phenylphosphate and β-glycerophosphate, respectively. Vmax was also a function of pH with and without the inhibitor. Rat intestinal alkaline phosphatase exhibited maximum enzyme activity at pH 9.8 and 8.8 with substrates, phenylphosphate and β-glycerophosphate, respectively, in presence of the noninhibitor, d-phenylalanine. The corresponding pH optima in the presence of l-phenylalanine inhibitor were 10.2 and 9.3, respectively. This shift in optimum pH by the inhibitor was observed in systems containing carbonate-bicarbonate or borate buffers. The Michaelis constant was pH-dependent. The Dixon plot (pKm with respect to pH) showed one discontinuity at pH 8.6 for the free enzyme and another at pH 9.6 for the enzyme-phenylphosphate complex. The values for the energy of activation for the enzyme-catalyzed hydrolysis of phenylphosphate with and without l-phenylalanine were 18,000 and 6,000 calories per mole, respectively. The inhibition was greatly dependent on substrate and inhibitor concentrations, and was of the uncompetitive type, because the double reciprocal plots of velocity and substrate concentrations in the presence of four different concentrations of l-phenylalanine were all straight lines parallel to those obtained without the inhibitor, in both the cases of phenylphosphate and β-glycerophosphate. At this time, the kinetic data are interpreted as indicating either the formation of a thermodynamically stable enzyme-inhibitor-substrate complex which, in effect, reduces the concentration of enzyme-substrate complex available to decompose into products or the production of a weakly dissociable enzyme-inhibitor-substrate complex. These interpretations are relevant to the explanation of the stereo-specific, organ-specific inhibition of rat intestinal alkaline phosphatase by l-phenylalanine.
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