Suzuki J, Sasaki K, Sasao Y, Hamu A, Kawasaki H, Nishiyama M, Horinouchi S, Beppu T
Department of Agricultural Chemistry, Faculty of Agriculture, University of Tokyo, Japan.
Protein Eng. 1989 May;2(7):563-9. doi: 10.1093/protein/2.7.563.
Artificial mutations of chymosin by recombinant DNA techniques were generated to analyze the structure--function relationship in this characteristic aspartic proteinase. In order to prepare the mutant enzymes in their active form, we established procedures for purification of correctly refolded prochymosin from inclusion bodies produced in Escherichia coli transformants and for its subsequent activation. Mutagenesis by linker insertion into cDNA produced several mutants with an altered ratio of milk clotting activity to proteolytic activity and a different extent of stability. In addition to these mutants, several mutants with a single amino acid exchange were also constructed by site-directed mutagenesis and kinetic parameters of these mutant enzymes were determined by using synthetic hexa- and octa-peptides as substrates. Exchange of Tyr75 on the flap of the enzyme to Phe caused a marked change of substrate specificity due to the change of kcat or Km, depending on the substrate used. Exchange of Val110 and Phe111 also caused a change of kinetic parameters, which indicates functional involvement of these hydrophobic residues in both the catalytic function and substrate binding. The mutant Lys220----Leu showed a marked shift of the optimum pH to the acidic side for hydrolysis of acid-denatured haemoglobin along with a distinct increase in kcat for the octa-peptide in a wide pH range.
通过重组DNA技术对凝乳酶进行人工突变，以分析这种典型天冬氨酸蛋白酶的结构-功能关系。为了制备具有活性形式的突变酶，我们建立了从大肠杆菌转化体产生的包涵体中纯化正确重折叠的凝乳酶原的程序及其随后的激活程序。通过将接头插入cDNA进行诱变产生了几种突变体，其凝乳活性与蛋白水解活性的比率发生了改变，稳定性程度也不同。除了这些突变体，还通过定点诱变构建了几个单氨基酸交换的突变体，并使用合成六肽和八肽作为底物测定了这些突变酶的动力学参数。酶的瓣上的Tyr75被Phe取代，由于kcat或Km的变化，根据所使用的底物不同，导致底物特异性发生明显变化。Val110和Phe111的交换也导致动力学参数的变化，这表明这些疏水残基在催化功能和底物结合中都发挥作用。突变体Lys220→Leu在广泛的pH范围内，对于酸变性血红蛋白的水解，最佳pH向酸性侧明显偏移，同时八肽的kcat明显增加。
