OXIDATIVE MODULATION AND INACTIVATION OF RABBIT CARDIAC ADENYLATE DEAMINASE

Oxidative stress and adenine nucleotide catabolism occur concomitantly in several disease states, such as cardiac ischaemia-reperfusion, and may act as synergistic determinants of tissue injury. However, the mechanisms underlying this potential interaction remain ill-defined. We examined the influence of oxidative stress on the molecular, kinetic and regulatory properties of a ubiquitous AMP-catabolizing enzyme, adenylate deaminase (AMPD) (EC 3.5.4.6). To this intent, rabbit heart AMPD and an H2O2/ascorbate/iron oxidation system were employed. Enzyme exposure to the complete oxidation system acutely impaired its catalytic activity, lowered the V-max by 7-fold within 5 min, and rendered the enzyme unresponsive to nucleotide effecters. Irreversible AMPD inactivation resulted within about 15 min of oxidative insult and was not prevented by free-radical scavengers. Oxidative stress did not affect the molecular mass, tetrameric nature, K-m, immunoreactivity or trypsinolytic pattern of the enzyme; nor did it induce carbonyl formation, Zn2+ release from the holoenzyme or net AMPD S-thiolation. This injury pattern is inconsistent with a radical-fragmentation mechanism as the basis for the oxidative AMPD inactivation observed. Rather, the sensitivity of the enzyme to both S-thiolation and thiol alkylation and the significant (3 of 9/mol of denatured enzyme) net loss of DTNB-reactive thiols on exposure to oxidant strongly implicate the conversion of essential thiol moieties into stable higher-oxidation states in the oxidative inactivation of cardiac AMPD. The altered thiol status of the enzyme on oxidative insult may prohibit a catalytically permissible conformation and, in so doing, increase AMP availability to 5'-nucleotidase in vivo.