Kikuchi M, Yamamoto Y, Taniyama Y, Ishimaru K, Yoshikawa W, Kaisho Y, Ikehara M
Protein Engineering Research Institute, Osaka, Japan.
Proc Natl Acad Sci U S A. 1988 Dec;85(24):9411-5. doi: 10.1073/pnas.85.24.9411.
Computer graphics indicate that a steric hindrance exists between valine-110 side chain of human lysozyme (EC 3.2.1.17) and an acetyl group of a modified substrate that contains N6,O-diacetylmuramic acid. To alter the substrate specificity of human lysozyme to be effective on the modified substrate, we replaced the valine-110 residue with various amino acids by site-directed mutagenesis. One of the mutant proteins (valine residue replaced with proline:P110) was secreted in Saccharomyces cerevisiae as at least four components (P110-A, P110-B, P110-C, and P110-D) with different specific activities. Two components, P110-B and P110-D, were isolated in a pure form and structurally characterized. The results suggest that this mutation lowered the lytic activity against Micrococcus lysodeikticus by changing a local conformation of the catalytic site while keeping almost the same substrate binding sites. Our results also indicate that cis/trans isomerization of prolyl peptide bonds probably occurs in vivo and that the conformational change of protein as well as point mutations in genes might influence the molecular evolution of the protein.
计算机图形学表明，人溶菌酶（EC 3.2.1.17）的缬氨酸-110侧链与含有N6,O-二乙酰胞壁酸的修饰底物的乙酰基之间存在空间位阻。为了改变人溶菌酶的底物特异性以使其对修饰底物有效，我们通过定点诱变将缬氨酸-110残基替换为各种氨基酸。其中一种突变蛋白（缬氨酸残基被脯氨酸取代：P110）在酿酒酵母中分泌为至少四种具有不同比活性的组分（P110-A、P110-B、P110-C和P110-D）。两种组分P110-B和P110-D被纯化并进行了结构表征。结果表明，这种突变通过改变催化位点的局部构象同时保持几乎相同的底物结合位点，降低了对溶壁微球菌的裂解活性。我们的结果还表明，脯氨酰肽键的顺/反异构化可能在体内发生，并且蛋白质的构象变化以及基因中的点突变可能影响蛋白质的分子进化。
