Rapid determination of adenosine deaminase kinetics using fast-scan cyclic voltammetry.
Adenosine deaminase is an enzyme involved in purine metabolism and its inhibitors are used as anticancer and antiviral drugs. In this study, we show that fast-scan cyclic voltammetry at carbon-fiber microelectrodes can be used to study the kinetics of adenosine deaminase by electrochemically monitoring decreases in adenosine concentration. Buffer and salt concentrations were shown to affect the enzyme kinetics and the inhibition by erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) and deoxycoformycin (DCF). In a Tris buffer containing salts that mimic cerebrospinal fluid, EHNA and DCF showed non-competitive inhibition with a K(i) of 1.7 +/- 0.6 nM and 1.2 +/- 0.2 nM, respectively. However, removing the divalent cations from the Tris buffer caused the inhibition to be competitive and reduced the K(i) for DCF by two orders of magnitude. In phosphate-buffered saline, the K(i) was 1.0 +/- 0.2 nM for EHNA and 3.6 +/- 0.3 pM for DCF, similar to literature values. Adenosine deaminase was also competitively inhibited by AgNO(3), showing it is susceptible to silver toxicity. Caffeine was found to increase adenosine deaminase activity. This is a fast, easy method for screening drug effects on enzyme kinetics and could be applied to other enzymatic reactions where there is a significant difference in the electroactivity of the reactant and product.
Xu
Yida
Y
Department of Chemistry, University of Virginia, P.O. Box 400319, Charlottesville, VA, USA.
Venton
B Jill
BJ
D000225:Adenine / Q000031:analogs & derivatives / Q000494:pharmacology; D000241:Adenosine; D000243:Adenosine Deaminase / Q000378:metabolism*; D058892:Adenosine Deaminase Inhibitors / Q000494:pharmacology; D000818:Animals; D002110:Caffeine / Q000494:pharmacology; D002417:Cattle; D004553:Electric Conductivity; D004563:Electrochemistry / Q000379:methods*; D057075:Enzyme Assays / Q000379:methods*; D007700:Kinetics; D010084:Oxidation-Reduction; D015649:Pentostatin / Q000494:pharmacology; D058915:Purinergic P1 Receptor Antagonists / Q000494:pharmacology; D018047:Receptors, Purinergic P1 / Q000378:metabolism; D012834:Silver / Q000494:pharmacology; D013997:Time Factors
D058892:Adenosine Deaminase Inhibitors; D058915:Purinergic P1 Receptor Antagonists; D018047:Receptors, Purinergic P1; D015649:Pentostatin; D002110:Caffeine; D012834:Silver; C010192:9-(2-hydroxy-3-nonyl)adenine; D000243:Adenosine Deaminase; D000225:Adenine; D000241:Adenosine