Expression and characterization of mutant forms of the type I regulatory subunit of cAMP-dependent protein kinase. The effect of defective cAMP binding on holoenzyme activation.

摘要: Abstract The mouse wild type and four mutant regulatory I (RI) subunits were expressed in Escherichia coli subjected to kinetic analyses. defective RI had point mutations either cAMP-binding site A (G200/E), B (G324/D, R332/H), or both binding sites. In addition, a truncated form of which lacked the entire was generated. All bound [3H]cAMP demonstrated more rapid rates cAMP dissociation compared subunit. Dissociation profiles showed only single component, suggesting that nonmutated functional. associated with purified native catalytic subunit chromatographically separable holoenzyme complexes activity suppressed. Each these holoenzymes could be activated but varying degrees responsiveness apparent Ka values ranging from 40 nM greater than 5 microM. extent mutated sites also shown by resistance respective activation analogs selective for Kinetic results support conclusions 1) Gly-200 Gly-324 Arg-332 are essential normal conformation function, 2) cAMP-dependent protein kinase requires one functional, 3) mutational inactivation (slow exchange) has much drastic effect on increasing cAMP, as well altering rate remaining free strong dependence integrity other suggests tight association between two