Kinetics and Mechanism of the Binding of Pyridoxal 5′-Phosphate to Apoglutamate Decarboxylase

• Published in: The Journal of biological chemistry 1972-11, Vol.247 (21), p.7097-7105
• Main Authors: O'Leary, Marion H., Malik, Joseph M.
• Format: Article
• Language: English
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)44698-X

The rate of binding of pyridoxal 5′-phosphate to apoglutamate decarboxylase from Escherichia coli can be measured by absorption spectroscopy, fluorescence, circular dichroism, and enzyme activity. All four methods give the same results under all conditions examined. At pH 4.9 in pyridinium chloride buffers the binding at a given coenzyme concentration follows first order kinetics. The rate is not first order in coenzyme, but instead levels off at coenzyme concentrations near 1 mm. A plot of the reciprocal of the observed first order rate constant versus the reciprocal of the coenzyme concentration is linear. The binding of 4-deoxypyridoxine 5′-phosphate, like that of pyridoxal 5′-phosphate, follows saturation kinetics, and thus involves at least two distinct steps. The dissociation constant for the first step (0.084 mm) and the rate constant for the second step (0.14 min-1) differ only slightly from those for pyridoxal 5′-phosphate under the same conditions (0.19 mm and 0.20 min-1, respectively). The pKa of the phenolic hydroxyl group of the deoxy compound is shifted downward by more than 3 units on binding to the enzyme. These data are used to derive a general mechanism for the binding of pyridoxal 5′-phosphate to glutamate decarboxylase. [see PDF for equation] Complex-I is a rapidly formed complex in which there is weak association between the phosphate of the coenzyme or analog and the protein. The interconversion of Complex-I and Complex-II is a conformation change of the protein and is rate-determining. The catalytically active Schiff base is formed in the last step. The kinetic constants in the above scheme vary according to the anion present. Much faster binding is observed in pyridinium chloride buffer than in pyridinium sulfate or acetate. Fluoride is also a potent a ctivator of the binding The rate constant for the binding of the coenzyme under optimum conditions is at least 100-fold smaller than that predicted from model reactions. In dilute solution and at higher pH the enzyme dissociates into subunits. Coenzyme binding to the dissociated enzyme is several times faster than binding to the aggregated enzyme. 3-Pyridinemethanol phosphate, benzyl phosphate, and inorganic phosphate inhibit the binding of pyridoxal 5′-phosphate to apoglutamate decarboxylase. The inhibition constants are 3.0, 25, and 40 mm, respectively, at pH 4.9. This indicates that the binding of the coenzyme to the protein involves ionic bonding to the phosphate and hydrogen bond