Evidence for species-specific substrate-site-directed inactivation of rabbit adenylate kinase by N6-(6-iodoacetamido-n-hexyl)adenosine 5'-triphosphate

Abstract

Adenosine 5'-triphosphate (ATP) derivatives bearing iodoacetylamino-n-alkyl substituents [(CH2)nNHCOCH2I] on N6 were synthesized as potential ATP-site-directed irreversible inhibitors of adenylate kinases from rabbit, pig, and carp muscle. When n was 5 no enzyme was progressively inhibited (inactivated) by 1 mM inhibitor under the test conditions (6 h at 0 degrees); when n was 6 the rabbit enzyme was 76% inactivated by 0.79 mM inhibitor whereas the pig and carp enzymes were unaffected by 2.76 mM inhibitor; when n was 7, 1 mM inhibitor inactivated 14% of the rabbit enzyme and did not inactivate the pig and carp enzymes; when n was 8, all enzymes were inactivated 11-15% by 1 mM inhibitor. No inactivation occurred when the iodine of the hexamethylene analogue was replaced by hydrogen. The selective effect occured also in mixtures of the rabbit and pig enzymes and evidence could not be found that the hexamethylene analogue was activated by the rabbit enzyme or deactivated by the pig and carp preparations. The species-specific inactivation in concluded from various lines of evidence to be ATP-site-directed and is attributed to alkylation of an amino acid residue of the rabbit enzyme which in the pig and carp enzymes is absent, inaccessible, or less reactive. These and previous studies with several other enzymes provide evidence that substrate-site-directed agents capable of bonding covalently to an amino acid residue outside the substrate site can be designed to exert species-specific or tissue-specific irreversible inhibition of target enzymes.