Magnetic resonance studies of the effect of the regulatory subunit on metal and substrate binding to the catalytic subunit of bovine heart protein kinase.
Magnetic resonance studies of the effect of the regulatory subunit on metal and substrate binding to the catalytic subunit of bovine heart protein kinase.
复制标题
磁共振研究调节亚基对与牛心蛋白激酶催化亚基结合的金属和底物的影响。
DOI：
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发表时间：
1980
影响因子：
4.8
通讯作者：
E. Kaiser
中科院分区：
文献类型：
--
作者：
J. Granot;A. Mildvan;K. Hiyama;H. Kondo;E. Kaiser
The binding of the regulatory subunit to the catalytic subunit of protein kinase is known to completely inhibit the catalytic activity by formation of the inactive holoenzyme. Magnetic resonance methods have been used to compare the binding of ADP, Mn2+, and a peptide substrate and substrate analog to the holoenzyme or to the catalytic subunit. ADP and MnADP are found to bind to the holoenzyme with a &fold greater affinity than to the catalytic subunit. The ADP complexes of both the holoenzyme and the catalytic subunit bind Mn2+ indistinguishably, at an activating site and an inhibitory site. Hence, inhibition by the regulatory subunit does not result from interference with the binding of the nucleotide substrate or its metal complex to the active site, nor does it result from enhanced binding of the divalent cation at a previously identified inhibitory site. The catalytic subunit of protein kinase enhances the effect of the bound Mn2+-nucleotide on l / T z of the side chain protons of the peptide substrate Leu-ArgArg-Ala-Ser-Leu-Gly and its competitive analog LeuArg-Arg-Ala-Ala-Leu-Gly. These effects are abolished by the competitive inhibitor, polyarginine, establishing active site binding. Using this enhancement of 1/Tz as an indicator of active site binding, the regulatory subunit was found to block such binding of the peptide substrate and its analog. The addition of a saturating level of CAMP which increases the dissociation constant of the holoenzyme -lo4-fold, however, does not dissociate the holoenzyme at the enzyme concentrations required by the NMR experiments (-20 p ~ ) . Phosphorylation of the regulatory subunit, which increases the dissociation constant of the holoenzyme by an order of magnitude, followed by the addition of CAMP, results in dissociation of the regulatory subunit, permitting the peptide substrate analog to bind to the active site of the catalytic subunit. From these observations, as well as from other known properties of protein kinase, a mechanism of regulation is proposed in which the regulatory subunit inhibits the enzyme by blocking the binding site of the protein substrate.
