CARDIAC ADENYLATE DEAMINASE - MOLECULAR, KINETIC AND REGULATORY PROPERTIES UNDER PHOSPHATE-FREE CONDITIONS

Adenylate deaminase (EC 3.5.4.6) may help to regulate the adenine nucl eotide catabolism characteristic of such disease states as myocardial ischaemia. We report analysis of the molecular, kinetic and allosteric properties of rabbit heart adenylate deaminase when extracted and pur ified under phosphate-free conditions (i.e., with Hepes/KOH). The enzy me's subunit molecular mass (similar to 81 kDa), pI (6.5), substrate s pecificity for 5'-AMP, and activation by K+ were identical in the abse nce or presence of phosphate. At each chromatographic step during isol ation without phosphate, cardiac adenylate deaminase showed a lower ap parent activity as compared with the enzyme prepared with phosphate pr esent. Kinetic constants for the phosphate-free rabbit heart adenylate deaminase preparation (K-m 0.54 mM AMP; V-max 1.4 mu mol/min per mg o f protein) were similar to 10-fold lower than those of the enzyme isol ated with phosphate. The same irreversible decrease in kinetic constan ts could be achieved by dialysing phosphate from the phosphate-contain ing enzyme preparation. The relationship between enzyme activity and s ubstrate concentration was sigmoidal in the presence of phosphate, but hyperbolic in its absence. Cardiac adenylate deaminase under phosphat e-free conditions was no longer allosterically activated by ATP and AD P, yet remained inhibitable by GTP. Enzyme inhibition by the transitio n-state mimic coformycin was not influenced by phosphate status. The p hosphate-free preparation of rabbit heart adenylate deaminase was mark edly labile and extremely susceptible to proteolysis by trypsin or chy motrypsin. The inactivation kinetics and fragmentation pattern in resp onse to controlled proteolysis depended on whether the enzyme had been isolated with or without phosphate present, suggesting a conformation al difference between the two enzyme preparations. These data constitu te direct evidence that the absence of phosphate irreversibly converts cardiac adenylate deaminase into a pseudoiso-enzyme with distinct kin etic, regulatory and stability properties.