Gibbs C S, Zoller M J
Department of Protein Engineering, Genentech Inc., South San Francisco, California 94080.
Biochemistry. 1991 Jun 4;30(22):5329-34. doi: 10.1021/bi00236a001.
"Charged-to alanine" scanning mutagenesis of the catalytic subunit of the Saccharomyces cerevisiae cAMP-dependent protein kinase (C1) identified three glutamate residues, E171, E214, and E274, that are involved in the recognition of a peptide substrate, kemptide (Leu1Arg2Arg3Ala4Ser5Leu6Gly7). These glutamate residues are conserved or conservatively substituted with asparate in the serine/threonine protein kinases that have a requirement for basic residues on the N-terminal side of their phosphorylation sites. Alanine replacement mutants in C1 were subjected to kinetic analysis using alanine-substituted peptides as substrates. The additivity or nonadditivity of the effects of the alanine substitutions on the catalytic efficiency (kcat/Km) was analyzed. This allowed the identification of electrostatic interactions between the three glutamate residues in the enzyme and the two arginine residues present in the peptide substrate. The data suggest that E171 interacts with Arg2 in the substrate and that E214 and E274 both interact with Arg3. This may be a general method for identifying simple intermolecular interactions involving proteins when there is no three-dimensional structure available of the complex of interacting species. The identification of these interactions provides the potential for rational protein engineering of enzymes with alternative specificities.
对酿酒酵母环磷酸腺苷依赖性蛋白激酶（C1）催化亚基进行“丙氨酸取代”扫描诱变，确定了三个谷氨酸残基E171、E214和E274，它们参与了肽底物肯普肽（Leu1Arg2Arg3Ala4Ser5Leu6Gly7）的识别。在其磷酸化位点N端侧需要碱性残基的丝氨酸/苏氨酸蛋白激酶中，这些谷氨酸残基保守存在或被天冬氨酸保守取代。使用丙氨酸取代的肽作为底物，对C1中的丙氨酸替代突变体进行动力学分析。分析了丙氨酸取代对催化效率（kcat/Km）影响的加和性或非加和性。这使得能够确定酶中的三个谷氨酸残基与肽底物中存在的两个精氨酸残基之间的静电相互作用。数据表明，E171与底物中的Arg2相互作用，E214和E274均与Arg3相互作用。当相互作用物种的复合物没有三维结构时，这可能是一种识别涉及蛋白质的简单分子间相互作用的通用方法。这些相互作用的确定为具有替代特异性的酶的合理蛋白质工程提供了可能性。
