Kinetics of the Binding of Pyridoxal 5'-Phosphate to Glutamate Decarboxylase

The rate of binding of pyridoxal 5'-phosphate (PLP) to apoglutamate decarboxylase has been measured as a function of coenzyme concentration at 20°, pH 4.9, by measuring the change in absorbance at 355 mµ and 427 mµ and by measuring enzyme activity versus time. All three measures give the same rate, and first order kinetics is observed for at least three half-lives. A plot of 1/ k obs versus 1/[PLP] gives an excellent straight line over a 20-fold range of PLP concentration. These results are interpreted in terms of a two-step mechanism involving rapid initial formation of an enzyme-PLP complex followed by slow formation of the catalytically active enzyme-PLP Schiff base. The rate of the second step is more than a 100-fold slower than the rate of the corresponding Schiff base formation involving PLP and n -butylamine.