L-amino-acid oxidase I. Effect of pH

Abstract

The effect of pH on K m and upon the rate constants associated with the reduction of oxidized enzyme by substrate and with the oxidation of reduced enzyme has been examined for the case of purified l-amino-acid oxidase ( l-amino-acid:O 2 oxidoreductase (deaminating), EC I.4.3.2) of Eastern Diamondback Rattlesnake ( Crotalus Adamanteus), using l-leucine as a substrate at 25°, 30° and 35°. The effect of pH on K m yields two p K's: one in the range 5.7–5.9 corresponding to the enzyme-substrate ( ES) intermediate and one in the range 8.2–8.4 corresponding to the free enzyme. The variation of the two sets of p K's with temperature both yield an apparent enthalpy of ionization of 7.5 kcal/mole. These data lead to the conclusion that histidyl is most likely involved in the catalysis at the active center. The variation of the reduction rate constant k obs with pH parallels the variation of K m with pH, indicating that the variation of k obs with pH depends on the variation in the availability of ES intermediate with pH. The oxidation rate constant, k ox, does not vary appreciably with pH, suggesting that the oxidation process proceeds via a direct combination of oxygen and reduced form or forms of the enzyme without the mediation of catalytic groups which may be titrated in the range of pH studied.