SUBSTRATE BINDING BY BLASTICIDIN-S DEAMINASE, AN AMINOHYDROLASE FOR NOVEL 4-AMINOPYRIMIDINE NUCLEOSIDES

The substrate specificity of blasticidin S deaminase (E.C. 3.5.4.23), purified from Aspergillus terreus, has been studied in detail. The enzyme was found to catalyze the hydrolytic deamination of cytosine nucleus in blasticidin S and its derivatives, while cytosine, cytidine, cytidine monophosphate, adenosine, and guanosine were not regarded as substrates. The structural requirements of compounds for binding by the enzyme were evaluated by the nature of inhibition and inhibitor constants; deaminohydroxyblasticidin S, the reaction product, and its derivatives inhibited the enzymatic aminohydrolysis of blasticidin S competitively. The Ki for deaminohydroxyblasticidin S was determined to be 2.3 .times. 10-5 M, which is close to the Km for blasticidin S (2.1 .times. 10-5 M). The binding affinity of derivatives to the enzyme, -.DELTA.Gbind, was calculated on the basis of Ki values, and further the partial binding affinities, -.DELTA.g, for several moieties or atomic groups in blasticidin S were deduced from the -.DELTA.Gbind values. The results showed that the enzyme involves a specific binding site with multiple points corresponding to the carboxyl group of cytosinine moiety, the amide group, and the .beta.-amino and guanidino groups of blastidic acid moiety in blasticidin S molecule; these facts are strongly indicative of the enzyme to be a new aminohydrolase for novel nucleosides such as blasticidin S.