Studies on Crystalline D-amino Acid Oxidase. I. Selective Inhibition in the Action of Sulfhydryl-binding Reagents.

In earlier work with partially purified preparations, we have examined inhibitory processes (l-3) and substrate specificity (4, 5) of n-amino acid oxidase. With availability of a highly purified crystallized enzyme prepared from hog kidney (6), an investigation of certain aspects of enzymatic mechanism has been initiated. In the present study enzymatic activity was observed to be depressed as a linear function of the concentration of any one of a number of sulfhydryl-binding reagents. The evaluation of such data is simplified when an enzyme contains one sulfhydryl group, but complexities arise when an enzyme, such as n-amino acid oxidase, contains multiple sulfhydryl groups capable of reacting with the inhibitor. Linear activity titrations have already been observed with several other polysulfhydryl enzymes (7-l 1) , but application of the results to possible elucidation of the role of the sulfhydryl group has been complicated by difficulty in the interpretation of the inhibition data on a molecular level. Certain properties of the n-amino acid oxidase have allowed us to study this phenomenon in detail and to conclude that upon admixture of enzyme and a limiting amount of inhibitor, a fraction of the enzyme molecules, in effect serially, bind a sufficient complement of inhibitor to become completely inactivated. Thus, when enough inhibitor is added to produce over-all 50% inactivation, one-half of the enzyme molecules are found to be inactive and to contain all added inhibitor; the remaining enzyme molecules retain full specific activity and are devoid of bound inhibitor. Certain parameters of this inhibitory process, such as the rate of the reaction between enzyme and inhibitor and the amount of inhibitor required for complete inhibition, were found to vary as a function of the state of oxidation and other attributes of the flavoenzyme. The implications and possible mechanisms for selective inhibition are discussed.