Granot J, Mildvan A S, Bramson H N, Kaiser E T
Biochemistry. 1980 Jul 22;19(15):3537-43. doi: 10.1021/bi00556a019.
Co3+ and Cr3+ complexes of beta, gamma-methylene-ATP (AMPPCP), which are substitution-inert substrate analogues inactive in phosphoryl transfer reactions, have been used in binding and structural studies of cAMP-dependent protein kinase. Dissociation constants of enzyme complexes with Co(NH3)4AMPPCP and CrAMPPCP and with Mn2+, which binds at an inhibitory site, were determined by electron paramagnetic resonance and by proton relaxation rate enhancement techniques. Nuclear relaxation rate measurements at 100 and 360 MHz were used to determine the distance between Mn2+ and the beta, gamma-methylene protons of Co-(NH3)4AMPPCP, yielding 7.4 +/- 0.6 A in the absence of enzyme and 5.0 +/- 0.9 A when both Mn2+ and Co-(NH3)4AMPPCP were bound to the enzyme. The effect of the paramagnetic CrAMPPCP on the electron spin relaxation time of the enzyme-bound Mn2+ was used used to calculate the distance between the two metal ions of 4.8 +/- 0.4 A. This distance and the Mn2+-methylene distance are consistent with the previous finding that the inhibitory metal bridges the enzyme to the triphosphate chain of the enzyme-bound nucleotide [Granot, J., Kondo, H., Armstrong, R. M., Mildvan, A. S., & Kaiser, E. T. (1979) Biochemistry 18, 2339]. From the paramagnetic effects on the relaxation rates of the protons of the peptide substrate Leu-Arg-Arg-Ala-Ser-Leu-Gly, distances from Mn2+ and Cr3+ to the serine methylene protons of 9.1 +/- 0.9 and 8.1 +/- 0.8 A, respectively, were calculated. These and previous measurements were used to estimate a distance of 5.3 +/- 0.7 A along the reaction coordinate between the gamma-phosphorus of ATP and the serine hydroxyl oxygen. This distance is 2 A greater than that required for molecular contact. The mechanistic implications of these findings are discussed.
β,γ-亚甲基-ATP(AMPPCP)的Co3+和Cr3+配合物是在磷酸转移反应中无活性的取代惰性底物类似物,已被用于cAMP依赖性蛋白激酶的结合和结构研究。通过电子顺磁共振和质子弛豫率增强技术测定了酶与Co(NH3)4AMPPCP和CrAMPPCP以及与在抑制位点结合的Mn2+形成的复合物的解离常数。利用100和360 MHz下的核弛豫率测量来确定Mn2+与Co-(NH3)4AMPPCP的β,γ-亚甲基质子之间的距离,在没有酶的情况下为7.4±0.6 Å,当Mn2+和Co-(NH3)4AMPPCP都与酶结合时为5.0±0.9 Å。顺磁性CrAMPPCP对酶结合的Mn2+的电子自旋弛豫时间的影响被用于计算两个金属离子之间的距离为4.8±0.4 Å。这个距离和Mn2+ - 亚甲基距离与之前的发现一致,即抑制性金属将酶与酶结合核苷酸的三磷酸链连接起来[格拉诺特,J.,近藤,H.,阿姆斯特朗,R. M.,米尔德万,A. S.,& 凯泽,E. T.(1979年)《生物化学》18,2339]。根据对肽底物Leu-Arg-Arg-Ala-Ser-Leu-Gly质子弛豫率的顺磁效应,分别计算出Mn2+和Cr3+到丝氨酸亚甲基质子的距离为9.1±0.9 Å和8.1±0.8 Å。这些以及之前的测量结果被用于估计沿着反应坐标在ATP的γ-磷和丝氨酸羟基氧之间的距离为5.3±0.7 Å。这个距离比分子接触所需的距离大2 Å。讨论了这些发现的机制意义。