The ATP substrate site of a cyclic-nucleotide-independent protein kinase from porcine liver nuclei.
The ATP substrate site of a second messenger-independent protein kinase of the type NII from porcine liver nuclei was mapped using a series of 30 ATP derivatives with modifications at the base, ribose or triphosphate moiety. Ki values for these derivatives were determined by competition with [gamma-32P]ATP; they range from 4 microM to 1.5 mM. For a comparison with data previously reported for the catalytic subunit of cAMP-dependent protein kinase I from rabbit skeletal muscle, the Ki values were transformed into delta delta values. These values are related to the Ki value of unsubstituted ATP and indicate the decrease of affinity caused by the different substitutions. With both enzymes the major binding affinity is derived from the interaction of the adenine base. The contributions of the two ribosyl OH groups are marginal and the triphosphate moiety interacts most strongly with its beta-phosphoryl group. Between the two enzymes the most striking differences, however, were observed for the specificity of the nucleobase interaction. While an unmodified N-6 amino group is required in the case of the cAMP-dependent protein kinase, the nuclear enzyme seems to tolerate extensive modification at this position, such as the introduction of a keto group or a bulky benzyl residue. Obviously, the ATP site of the nuclear kinase has an open cleft next to the N-6 of the adenine and binding of the adenine occurs by hydrophobic interaction without the formation of hydrogen bonds to any of the adenine nitrogens.
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D002467:Cell Nucleus / Q000201:enzymology*; D000242:Cyclic AMP / Q000494:pharmacology; D008099:Liver / Q000201:enzymology*; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism*; D013379:Substrate Specificity; D013552:Swine
D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D011494:Protein Kinases