Ullmann D, Jakubke H D
Department of Biochemistry, University of Leipzig, Germany.
Eur J Biochem. 1994 Aug 1;223(3):865-72. doi: 10.1111/j.1432-1033.1994.tb19063.x.
The S' subsite specificity of clostripain from Clostridium histolyticum was investigated by acyl transfer to libraries of amino acid amides, Ala-Xaa dipeptides, proline derivatives and pentapeptides using N alpha-benzoyl-L-arginine ethyl ester as acyl donor. A pentapeptide library consisting of 29 pentapeptides with general structure Xaa-Ala-Ala-Ala-Gly, Ala-Xaa-Ala-Ala-Gly and Ala-Ala-Xaa-Ala-Gly, where Xaa represents Gly, Ala, Pro, Leu, Phe, Asp, Glu, Arg and Lys, was prepared by solid-phase synthesis. The data analysis was performed by HPLC and evaluated by statistical algorithms. The nucleophile efficiency covers a range of more than three orders of magnitude. In the P'1 position, low specificity for amino acid amides and Xaa-(Ala)3-Gly peptides was found, however, in the P'2 position, positively charged amino acid residues are strongly preferred. The negatively charged side chains of aspartic acid and glutamic acid in the P'1 and P'2 positions, respectively, show only poor nucleophilic behaviour. In the case of these amino acids, the S'-P' interactions depend significantly on their position of these residues in the peptide chain of the nucleophile. The transfer of aspartic acid and glutamic acid from P'1-P'3 increases the nucleophile efficiency by approximately two orders of magnitude. The aromatic side chains are not well accepted, especially in the case of P'3Phe. Surprisingly, P'1Gly leads to effective P'-S' interactions. However, the opposite result was obtained for P'2Gly. The high efficiency for Gly-NH2 does not fit with the hydrophobicity structure/activity relationships. In most cases, peptide chain elongation does not improve the nucleophile efficiency. The effective interaction of D-Leu-NH2 with the S' subsite of clostripain emphasizes the fact that the nucleophile stereospecificity is not restricted to L-amino acids. The results with proline derivatives indicate remarkably different specificities of the S' binding site which can only be explained by conformational restraints. A positive cooperativity between P'1Pro and P'2Gly and a negative cooperativity between P'1Pro and P'2Phe was observed. The arrangement of three proline residues next to each other represents a favourable conformation for effective enzyme-nucleophile interactions.
以Nα-苯甲酰-L-精氨酸乙酯作为酰基供体，通过将酰基转移至氨基酸酰胺、丙氨酸-氨基酸二肽、脯氨酸衍生物和五肽文库，对溶组织梭菌的梭菌蛋白酶的S'亚位点特异性进行了研究。通过固相合成制备了一个由29个五肽组成的五肽文库，其一般结构为Xaa-Ala-Ala-Ala-Gly、Ala-Xaa-Ala-Ala-Gly和Ala-Ala-Xaa-Ala-Gly，其中Xaa代表甘氨酸、丙氨酸、脯氨酸、亮氨酸、苯丙氨酸、天冬氨酸、谷氨酸、精氨酸和赖氨酸。通过高效液相色谱进行数据分析，并采用统计算法进行评估。亲核试剂效率涵盖超过三个数量级的范围。在P'1位置，发现对氨基酸酰胺和Xaa-(Ala)3-Gly肽的特异性较低，然而，在P'2位置，带正电荷的氨基酸残基是强烈优选的。分别位于P'1和P'2位置的天冬氨酸和谷氨酸的带负电荷侧链仅表现出较差的亲核行为。对于这些氨基酸，S'-P'相互作用显著取决于它们在亲核试剂肽链中的位置。从天冬氨酸和谷氨酸从P'1-P'3的转移使亲核试剂效率提高了约两个数量级。芳香族侧链不太容易被接受，特别是在P'3苯丙氨酸的情况下。令人惊讶的是，P'1甘氨酸导致有效的P'-S'相互作用。然而，对于P'2甘氨酸却得到了相反的结果。甘氨酸-NH2的高效率不符合疏水性结构/活性关系。在大多数情况下，肽链延长并不能提高亲核试剂效率。D-亮氨酸-NH2与梭菌蛋白酶的S'亚位点的有效相互作用强调了亲核试剂立体特异性不限于L-氨基酸这一事实。脯氨酸衍生物的结果表明S'结合位点具有明显不同的特异性，这只能通过构象限制来解释。观察到P'1脯氨酸和P'2甘氨酸之间存在正协同作用，而P'1脯氨酸和P'2苯丙氨酸之间存在负协同作用。三个脯氨酸残基彼此相邻的排列代表了有效的酶-亲核试剂相互作用的有利构象。
