Active site specificity of the adenylate cyclase catalytic unit from bovine caudate nucleus.
ATP analogues were used to study the active site specificity of the catalytic unit (C) of solubilized and partially purified bovine brain caudate nucleus adenylate cyclase. Phenylenediamine ATP (PD-ATP), 8-azido ATP (8-N3ATP), chromium(III) 3'-beta-alanylarylazido ATP (CrATPa), and 2',3'-dialdehyde ATP (oATP) are competitive inhibitors of C in the presence of the substrate MnATP and the activator forskolin. (Km for MnATP is 50 +/- 11 microM, n = 13). The Ki values determined under initial velocity conditions are: PD-ATP, Ki = 695 +/- 60 microM, n = 5; 8-N3ATP, Ki = 155 +/- 23 microM, n = 5; CrATPa, Ki = 7 +/- 3 microM, n = 2; oATP, Ki = 42 +/- 5 microM, n = 3. Irradiation of 100 microM 8-N3ATP by UV light (254 nm) causes the first-order loss of reagent either in the presence or absence of C. Concomitant irreversible inhibition of C in the presence of 8-N3ATP was more complex and asymptotically approached 50% within 4-6 min. Loss of C activity in controls was 10-20%. The fraction of C covalently modified by 8-N3ATP, alpha, was calculated for each time point of irradiation for an increasing initial concentration ([A]o) of 8-N3ATP. Extrapolated to infinite time of photolysis, the value of alpha reached a final level, termed alpha t whose magnitude depended on [A]o. From these data we calculated an apparent KD of 4.5 microM for 8-N3ATP. ATP protected against the irreversible inhibition due to 8-N3ATP. These data are most consistent with a mechanism of photoaffinity labeling involving equilibrium binding and covalent insertion of 8-N3ATP into the active site. These results indicate that the active site binds analogues of ATP which are considerably modified in the adenine, ribose, and gamma-phosphate portions and that the affinity of C for these analogues is within an order of magnitude of the Km for ATP.
J W
JW
Winslow
E J
EJ
Neer
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000494:pharmacology; D000262:Adenylyl Cyclases / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002421:Caudate Nucleus / Q000201:enzymology*; D002540:Cerebral Cortex / Q000201:enzymology; D007202:Indicators and Reagents; D007700:Kinetics; D046911:Macromolecular Substances; D008433:Mathematics; D008954:Models, Biological; D010777:Photochemistry; D013329:Structure-Activity Relationship
D007202:Indicators and Reagents; D046911:Macromolecular Substances; D000255:Adenosine Triphosphate; D000262:Adenylyl Cyclases