Mutagenesis of human adenosine deaminase to active forms that partially resist inhibition by pentostatin

Abstract

Human adenosine deaminase (ADA) cDNA was randomly mutagenized in vitro and bacterial transformants were selected for resistance to the potent enzyme inhibitor, pentostatin (dCF). Cells transformed with mutant plasmids dCF-R2 and dCF-R6 were able to grow in the presence of 10(-6) M dCF, whereas 10(-11) M dCF blocked growth of cells complemented with wild-type ADA. DNA sequence analysis revealed double G/A conversions mutating Lys11 and Gln255 in dCF-R2, and Gly 31 and Glu 99 in dCF-R6, to different amino acids. Located far from the enzyme active site, these substitutions did not greatly affect the Km or Ki of the enzyme but were predicted to destabilize interactions within the enzyme structure. Mutants such as these may be useful to analysis of the stereology of inhibitor binding to ADA and toxicity of dCF.