Substrate Binding by Adenosine Deaminase: SPECIFICITY, pH DEPENDENCE, AND COMPETITION BY MERCURIALS

Abstract

Takadiastase adenosine deaminase, purified of phosphoesterase contaminants, is found to catalyze deamination of free adenine, adenine nucleotides and oligonucleotides, and a variety of substituted adenosine and deoxyadenosine derivatives. 3‘- and 5‘-Adenosine monophosphates are bound only as monoanions. Activity on adenylic acid oligonucleotides decreases with increasing chain length, becoming negligible in long polymers. The Km for adenosine (but notVmax) varies with pH, indicating the presence of prototropic equilibria in both free substrate and free enzyme. In the pH range from 3 to 9.5, a single enzyme ionization (apparent pKa 8.9) is indicated as important for substrate binding. The enzyme resists inactivation by many reagents for ionizable side chains of amino acids, but is uniquely sensitive to inhibition by mercuric ion and several organic mercurials. Mercurial inhibition is reversible and strictly competitive with a variety of purine substrates and analogues, and is not the result of direct interaction of inhibitor with substrate. Involvement of sulfhydryl groups in binding of mercurials is indicated by spectrophotometric detection of mercaptide formation between p-mercuribenzoate and enzyme, which indicates a binding affinity in agreement with that determined from the kinetics of inhibition. Competitive inhibition by mercurials is also observed for adenosine deaminase from calf intestinal mucosa and cytidine deaminase from Escherichia coli.