Lu W, Apostol I, Qasim M A, Warne N, Wynn R, Zhang W L, Anderson S, Chiang Y W, Ogin E, Rothberg I, Ryan K, Laskowski M
Department of Chemistry, Purdue University, West Lafayette, IN 47907, USA.
J Mol Biol. 1997 Feb 21;266(2):441-61. doi: 10.1006/jmbi.1996.0781.
The P1 or primary specificity residue of standard mechanism canonical protein inhibitors of serine proteinases, inserts into the S1 primary specificity cavity of the cognate enzyme upon enzyme-inhibitor complex formation. Both natural evolution and protein engineering often change the P1 residue to greatly alter the specificity and the binding strength. To systematize such results we have obtained all 20 coded P1 variants of one such inhibitor, turkey ovomucoid third domain, by recombinant DNA technology. The variants were extensively characterized. The association equilibrium constants were measured at pH 8.30, 21 (+/-2) degrees C, for interaction of these variants with six well characterized serine proteinases with hydrophobic S1, cavities. The enzyme names are followed by the best, worst and most specific coded residue for each. Bovine chymotrypsin A alpha (Tyr, Pro, Trp), porcine pancreatic elastase (Leu/Ala, Arg, Ala), subtilisin Carlsberg (Cys, Pro, Glu), Streptomyces griseus proteinase A (Cys, Pro, Leu) and B (Cys, Pro, Lys) and human leukocyte elastase (Ile, Asp, Ile). The data set was merged with Ka values for five non-coded variants at P1 of turkey ovomucoid third domain obtained in our laboratory by enzymatic semisynthesis. The ratios of the highest to the lowest Ka for each of the six enzymes range from 10(6) to 10(8). The dominant force for binding to these pockets is the hydrophobic interaction. Excess steric bulk (too large for the pocket), awkward shape (Pro, Val and Ile), polarity (Ser) oppose interaction. Ionic charges, especially negative charges on Glu- and Asp- are strongly unfavorable. The Pearson pro duct moment correlations for all the 15 enzyme pairs were calculated. We suggest that these may serve as a quantitative description of the specificity of the enzymes at P1. The sets of Streptomyces griseus proteinases A and B and of the two elastases are strongly positively correlated. Strikingly, chymotrypsin and pancreatic elastase are negatively correlated (-0.10). Such correlations can be usefully extended to many other enzymes and to many other binding pockets to provide a general measure of pocket binding specificity.
丝氨酸蛋白酶标准机制典型蛋白抑制剂的P1或一级特异性残基，在酶-抑制剂复合物形成时插入同源酶的S1一级特异性腔中。自然进化和蛋白质工程常常改变P1残基，以极大地改变特异性和结合强度。为了使这些结果系统化，我们通过重组DNA技术获得了一种这样的抑制剂——火鸡卵类粘蛋白第三结构域的所有20种编码的P1变体。对这些变体进行了广泛的表征。在pH 8.30、21（±2）℃下，测量了这些变体与六种具有疏水S1腔且特征明确的丝氨酸蛋白酶相互作用的缔合平衡常数。每种酶后面依次列出最佳、最差和最具特异性的编码残基。牛胰凝乳蛋白酶Aα（酪氨酸、脯氨酸、色氨酸）、猪胰弹性蛋白酶（亮氨酸/丙氨酸、精氨酸、丙氨酸）、嗜热栖热菌蛋白酶（半胱氨酸、脯氨酸、谷氨酸）、灰色链霉菌蛋白酶A（半胱氨酸、脯氨酸、亮氨酸）和B（半胱氨酸、脯氨酸、赖氨酸）以及人白细胞弹性蛋白酶（异亮氨酸、天冬氨酸、异亮氨酸）。该数据集与我们实验室通过酶促半合成获得的火鸡卵类粘蛋白第三结构域P1处五个非编码变体的Ka值合并。六种酶中每种酶的最高Ka与最低Ka之比在10^6到10^8之间。与这些口袋结合的主要作用力是疏水相互作用。空间体积过大（对于口袋来说太大）、形状不合适（脯氨酸、缬氨酸和异亮氨酸）、极性（丝氨酸）会阻碍相互作用。离子电荷，尤其是谷氨酸和天冬氨酸上的负电荷非常不利。计算了所有15对酶的皮尔逊积矩相关性。我们认为这些可能作为对酶在P1处特异性的定量描述。灰色链霉菌蛋白酶A和B以及两种弹性蛋白酶的相关性很强且为正相关。引人注目的是，胰凝乳蛋白酶和胰弹性蛋白酶呈负相关（-0.10）。这种相关性可以有效地扩展到许多其他酶和许多其他结合口袋，以提供口袋结合特异性的一般度量。
