Adenosine cyclic 3',5'-monophosphate dependent protein kinase: a new fluorescence displacement titration technique for characterizing the nucleotide binding site on the catalytic subunit.
We determined the dissociation constant (Kd) of a series of nucleotides for the bovine skeletal muscle type II catalytic subunit by displacing lin-benzoadenosine 5'-diphosphate (lin-benzo-ADP) with increasing concentrations of competing nucleotide. The Kd of each nucleotide was calculated from the decreases in the fluorescence polarization of lin-benzo-ADP that accompany its displacement from the catalytic subunit. We found that modifications of the adenine moiety reduce nucleotide affinity for the enzyme. The effect was most pronounced with modifications at position 6 of the base. Replacement of the 3'-hydroxyl group of ribose with a hydrogen increased the affinity of the nucleotide; addition of phosphate to the 2'- or 3'-hydroxyl groups, on the other hand, decreased nucleotide affinity. MgATP and MgADP exhibited Kd's of about 10 microM. AMP, which contains a negatively charged alpha-phosphate, bound with reduced affinity (643 microM). Adenosine, which lacks a charged alpha-phosphate, bound with a higher affinity (32 microM). To learn more about the nature of the alpha-phosphate binding site, a series of uncharged and positively charged derivatives of the 5'-position on the ribose moiety was prepared. The uncharged derivatives bound with much greater affinity than the negatively charged AMP. The Kd's for 5'-tosyladenosine and 5'-iodo-5'-deoxyadenosine were 30 and 32 microM, respectively. Like the negatively charged AMP, positively charged derivatives also bound less tenaciously than the neutral species. The positively charged 5'-amino-5'deoxyadenosine, for example, exhibited a 15-fold higher Kd (506 microM) than the neutral congenors.(ABSTRACT TRUNCATED AT 250 WORDS)
D000345:Affinity Labels; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D000242:Cyclic AMP / Q000494:pharmacology; D005454:Fluorescence Polarization; D066298:In Vitro Techniques; D008670:Metals / Q000494:pharmacology; D009132:Muscles / Q000201:enzymology; D009711:Nucleotides; D011494:Protein Kinases / Q000378:metabolism*
D000345:Affinity Labels; D008670:Metals; D009711:Nucleotides; D000242:Cyclic AMP; D011494:Protein Kinases