Bhatnagar D, Glass D B, Roskoski R, Lessor R A, Leonard N J
Biochemistry. 1985 Feb 26;24(5):1122-9. doi: 10.1021/bi00326a009.
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.
在可磷酸化肽[[Ala34]组蛋白H2B-(29 - 35)]存在的情况下使用活化的环鸟苷酸依赖性蛋白激酶，我们发现线性苯并腺苷5'-二磷酸(lin-benzo-ADP)是该酶相对于ATP的竞争性抑制剂，其抑制常数Ki(22微摩尔)与通过荧光偏振滴定法测定的解离常数Kd(20微摩尔)相似。然而，在不存在可磷酸化肽的情况下测定的lin-benzo-ADP的Kd仅为12微摩尔。ADP的结合亲和力较低(Ki = 169微摩尔; Kd = 114微摩尔)。以[Ala34]组蛋白H2B-(29 - 35)作为磷酸受体，lin-benzo-ATP的米氏常数Km为29微摩尔，ATP的Km为32微摩尔。然而，以lin-benzo-ATP作为底物时的最大反应速度Vmax仅为以ATP作为底物时的0.06%[0.00623±0.00035对11.1±0.17微摩尔/(分钟·毫克)]。lin-benzo-ADP与激酶的结合依赖于二价阳离子。荧光偏振显示，Mg2+、Mn2+、Co2+、Ni2+、Ca2+、Sr2+和Ba2+支持核苷酸与该酶的结合;然而，Ca2+、Sr2+和Ba2+不支持任何可测量的磷酸转移酶活性。介导磷酸转移酶活性的金属离子有效性的排序为Mg2+＞Ni2+＞Co2+＞Mn2+。尽管这些结果与用环腺苷酸依赖性蛋白激酶观察到的结果相似[哈特尔，F. T.，罗斯科斯基，R.，小罗斯科斯基，R.，罗森达尔，M. S.，& 伦纳德，N. J.(1983)生物化学22，2347]，但观察到以lin-benzo- ATP作为底物时的Vmax以及肽底物对核苷酸(lin-benzo-ADP和ADP)结合的影响存在主要差异。
