CHARACTERIZATION OF RECOMBINANT ESCHERICHIA-COLI 5'-METHYLTHIOADENOSINE S-ADENOSYLHOMOCYSTEINE NUCLEOSIDASE - ANALYSIS OF ENZYMATIC-ACTIVITY AND SUBSTRATE-SPECIFICITY/

Recombinant E. coli 5'-methylthioadenosine/S-adenosylhomocysteine nucl eosidase (EC 3.2.2.9) was used to study the potential for this enzyme to serve as a target for chemotherapeutic intervention. An examination of the parameters required for enzymatic activity indicate that the n ucleosidase functions over a broad range of pH and temperature, with a cidic conditions and temperatures of 37-45 degrees C being optimal. An alogs of 5'-methylthioadenosine and adenosine were assessed as potenti al enzyme inhibitors and to provide details regarding substrate specif icity and reaction mechanism. The 5'-arylthio analog, 5'-(b-nitropheny l)thioadenosine, was the most potent enzyme inhibitor studied, with a K-i of 20nM, A mutant of the nucleosidase lacking the first 8 amino ac ids was engineered to determine the contribution of these conserved re sidues reward enzyme specificity. The truncated enzyme exhibited a K-m [MTA] of 1.43 mu M, approximately 3 fold higher than the K-m reported for the full-length nucleosidase. (C) 1996 Academic Press, Inc.