Maurer K H, Markgraf M, Goddette D
COGNIS GmbH, Düsseldorf, Germany.
Adv Exp Med Biol. 1996;379:243-56. doi: 10.1007/978-1-4613-0319-0_26.
Three natural variants of subtilisin lentus could be differentiated by their amino acid sequence and their specific activity with low molecular weight peptide substrates of the type sAAPFpNA. The variants had amino acid exchanges in five, respective six positions of their amino acid sequence, four of which are located in the substrate loop of the enzyme (positions 92 - 102). Variants of one type of highly alkaline subtilisin (subtilisin 309) were made by site directed mutagenesis, each containing one of the corresponding amino acid exchanges. These intermediate forms were tested for activity, pH-dependence and substrate specificity. The changes in substrate affinity were relatively small for substrates with different amino acids as P1 residue. The differences in activity on peptide-substrates could be related primarily to a single amino acid substitution in the S4 substrate binding pocket in position 102. With substrate variations in the P3 amino acid residue, changes in k(cat) and K(m) revealed the importance of the charged amino acid exchanged between subtilisin 309 and BLAP. By these experiments an interaction of amino acid position 101 and the P3 residue of the substrate could be demonstrated. The substitution of two differently charged amino acids in the substrate binding region resulted in an unchanged pH-profile of the natural enzyme. With the single exchange intermediates differences in the pH-profile could be found, depending on the substrate tested: a characteristic change was observed with casein as substrate, no such change occurred with hemoglobin.
嗜热栖热芽孢杆菌蛋白酶的三种天然变体可以通过它们的氨基酸序列以及对sAAPFpNA类型的低分子量肽底物的比活性来区分。这些变体在其氨基酸序列的五个或六个位置上有氨基酸交换，其中四个位于酶的底物环中（92 - 102位）。通过定点诱变制备了一种高碱性枯草芽孢杆菌蛋白酶（枯草芽孢杆菌蛋白酶309）的变体，每个变体都包含一种相应的氨基酸交换。对这些中间形式进行了活性、pH依赖性和底物特异性测试。对于具有不同氨基酸作为P1残基的底物，底物亲和力的变化相对较小。肽底物活性的差异主要与102位S4底物结合口袋中的单个氨基酸取代有关。随着P3氨基酸残基处底物的变化，k(cat)和K(m)的变化揭示了枯草芽孢杆菌蛋白酶309和BLAP之间交换的带电荷氨基酸的重要性。通过这些实验，可以证明氨基酸101位与底物的P3残基之间存在相互作用。底物结合区域中两个带不同电荷的氨基酸的取代导致天然酶的pH曲线不变。对于单个交换中间体，根据所测试的底物，在pH曲线上可以发现差异：以酪蛋白为底物时观察到特征性变化，以血红蛋白为底物时则没有这种变化。
