Adenosine cyclic 3',5'-monophosphate dependent protein kinase: a new fluorescence displacement titration technique for characterizing the nucleotide binding site on the catalytic subunit

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Abstract
Kd of a series of nucleotides for the bovine skeletal muscle type II catalytic subunit were determined 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. Modifications of the adenine moeity reduce nucleotide affinty 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 of about 10 .mu.M. AMP, which contains a negatively charged .alpha.-phosphate, bound with reduced affinity (643 .mu.M). Adenosine, which lacks a charged .alpha.-phosphate, bound with a higher affinity (32 .mu.M). 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 for 5''-tosyladenosine 5''-iodo-5''-deoxyadenosine were 30 and 32 .mu.M, 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 .mu.M) than the neutral congenors. The enzyme''s site complementary to the .alpha.-phosphate may be hydrophobic in nature. Adding hydrophobic groups to the positive charges at the 5-position increased the binding affinity [Kd for 5''-(ethylamino)-, 5''-(diethylamino)-, 5''-(triethylammonium)- and 5''-(diallylamino)-5''-deoxyadenosine are 403, 284, 153 and 102 .mu.M, respectively]. The binding of lin-benzo-ADP to the catalytic subunit of protein kinase is dependent upon a divalent cation. Several metals were tested for their ability to promote binding and to support phosphotransferase activity. Fluorescence polarization studies revealed that Mg2+, Mn2+, Co2+, Cd2+, Ca2+ and Sr2+ supported nucleotide binding to the catalytic subunit, whereas Ba2+, Cr2+, Fe2+, Ni2+, Zn2+, Cu2+, Gd3+ and La3+ did not. Even though Ca2+ and Sr2+ promoted nucleotide binding, no measurable phosphotransferase activity was observed in their presence.

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