Interaction of guanosine cyclic 3',5'-phosphate dependent protein kinase with lin-benzoadenine nucleotides

Abstract

Using the activated cGMP-dependent protein kinase in the presence of the phosphorylatable peptide [[Ala34]histone H2B-(29-35)], we found that lin-benzoadenosine 5'-diphosphate (lin-benzo-ADP) was a competitive inhibitor of the enzyme with respect to ATP with a Ki (22 microM) similar to the Kd (20 microM) determined by fluorescence polarization titrations. The Kd for lin-benzo-ADP determined in the absence of the phosphorylatable peptide, however, was only 12 microM. ADP bound with lower affinity (Ki = 169 microM; Kd = 114 microM). With [Ala34]histone H2B-(29-35) as phosphoryl acceptor, the Km for lin-benzo-ATP was 29 microM, and that for ATP was 32 microM. The Vmax with lin-benzo-ATP, however, was only 0.06% of that with ATP as substrate [0.00623 +/- 0.00035 vs. 11.1 +/- 0.17 mumol (min.mg)-1]. Binding of lin-benzo-ADP to the kinase was dependent upon a divalent cation. Fluorescence polarization revealed that Mg2+, Mn2+, Co2+, Ni2+, Ca2+, Sr2+, and Ba2+ supported nucleotide binding to the enzyme; Ca2+, Sr2+, and Ba2+, however, did not support any measurable phosphotransferase activity. The rank order of metal ion effectiveness in mediating phosphotransferase activity was Mg2+ greater than Ni2+ greater than Co2+ greater than Mn2+. Although these results were similar to those observed with the cAMP-dependent protein kinase [Hartl, F. T., Roskoski, R., Jr., Rosendahl, M. S., & Leonard, N. J. (1983) Biochemistry 22, 2347], major differences in the Vmax with lin-benzo-ATP as substrate and the effect of peptide substrates on nucleotide (both lin-benzo-ADP and ADP) binding were observed.

Crystal Structures of Catalytic Subunit of cAMP-dependent Protein Kinase in Complex with Isoquinolinesulfonyl Protein Kinase Inhibitors H7, H8, and H89

“…On the other hand, only AMP, but not MgADP or MgATP, is able to replace lin-benzoadenosine from the modified kinase, indicating similar binding sites for adenosine, lin-benzoadenosine, and for H8, distinct from the ␤-and ␥-phosphate subsite, corresponding to the crystal structures. Slightly different behavior of lin-benzo-nucleotides has been observed in cGPK (39).…”

Synthetic peptide analogs differentially alter the binding affinities of cyclic nucleotide-dependent protein kinases for nucleotide substrates

“…(a) Dilution Titrations. Anisotropy as a function of varying enzyme concentration (at constant //«-benzo-ADP concentration) was measured as described earlier (Bhatnagar et al, 1985) to determine rb, which is the anisotropy value when all //«-benzo-ADP is bound to the protein kinase (i.e., at infinite enzyme concentration). The theoretical value of rb and the average angle of rotation for //«-benzo-ADP rigidly bound to the protein kinases were calculated from Perrin's equations as described earlier by using the value of r0 as 0.301 for //«benzo-ADP (Bhatnagar et al, 1985) and the following anisotropy relationship (Lakowicz, 1983):…”

“…MgSQ4 was then added to a final concentration of 10 mM, and the increased value of anisotropy, due to the binding of metal-nucleotide complex to the enzyme, was recorded (rmax). Anisotropy (z-obsd) was then recorded, as previously described (Bhatnagar et al, 1983(Bhatnagar et al, , 1985, at each nucleotide concentration after addition of successive increments (approximately 1-2 µ ) of //«-benzo-ADP to a constant protein kinase concentration, under the specified conditions of each experiment. " , and were calculated from Perrin's equations for anisotropy as described under Materials and Methods.…”

“…We have previously shown (Bhatnagar et al, 1983(Bhatnagar et al, , 1985 that the fluorescent "stretched-out" adenine nucleotide //«benzo-ADP is a competitive inhibitor with respect to ATP of both the catalytic subunit of cAMP-dependent protein kinase and the cGMP-dependent protein kinase. In the latter study, we also observed that the preferred phosphoryl-accepting substrate of cGMP-dependent protein kinase, the synthetic peptide [Ala34]histone H2B(29-35), decreased the affinity for both //«-benzo-ADP and ADP.…”

“…No such effect was observed with the cAMP-dependent enzyme. In this report we have used the fluorescent anisotropy titration technique (Bhatnagar et al, 1983(Bhatnagar et al, , 1985 to examine the extent to which the individual amino acid residues of this peptide substrate are involved in interactions with the enzymes that alter the affinity of nucleotide binding.…”