Williams H R, Lin T Y, Navia M A, Springer J P, Hoogsteen K
Biochem J. 1987 Feb 15;242(1):267-73. doi: 10.1042/bj2420267.
The binding constants of a number of ligands were measured for pancreatic elastase (PE) and anhydro-elastase (AE) in order to assess the contribution of Ser-195 to substrate and inhibitor binding by PE. AE was purified by affinity chromatography on a column containing immobilized turkey ovomucoid inhibitor. The AE had 0.1 +/- 0.1% of the activity of the native enzyme and contained 0.8 +/- 0.06 residue of dehydroalanine per molecule. A difference electron-density map, derived from an X-ray crystallographic analysis of AE, showed that the modified residue was Ser-195. The complexing of 3-carboxypropionyl-Ala-Ala-Ala-p-nitroanilide (SAN) to the active site of AE was also demonstrated by X-ray-diffraction analysis of an AE crystal soaked overnight with substrate. The nitroanilide moiety was not observed in the difference map. AE was shown to bind turkey ovomucoid inhibitor with a dissociation constant (Kd) of 0.3 +/- 0.06 microM compared with 0.10 microM for PE. The Kd of the AE-SAN complex (0.2 mM) was comparable with the Michaelis constant for SAN with PE (1.0 mM). A number of inhibitors, such as elastatinal, which forms a hemiketal adduct with PE, while others such as the beta-lactams, which function as acylators of the active-site serine residue, bound AE with a lower affinity than to PE. The binding of a peptidylchloromethane (acetyl-Ala-Ala-Pro-Ala-CH2Cl) to AE occurs without evidence for alkylation of histidine. The binding constants for benzoisothiazolinone and 3,4-dichloroisocoumarin to PE differed from their binding constants to AE by less than a factor of 4.0-fold. The contribution of the hydroxy group of Ser-195 to the binding of these inhibitors to PE in their non-covalent complexes is relatively small, even though they inactivate PE by an acylation mechanism. These results suggest that the hydroxy group on Ser-195 in PE is of secondary importance in the energetics of ligand binding, in contrast with its essential role in the catalytic properties of the enzyme.
为了评估丝氨酸-195对胰腺弹性蛋白酶（PE）底物和抑制剂结合的贡献，测定了多种配体与胰腺弹性蛋白酶（PE）和脱水弹性蛋白酶（AE）的结合常数。通过在含有固定化火鸡卵类粘蛋白抑制剂的柱上进行亲和层析来纯化AE。AE具有天然酶活性的0.1±0.1%，且每分子含有0.8±0.06个脱氢丙氨酸残基。源自AE的X射线晶体学分析的差分电子密度图表明，修饰的残基是丝氨酸-195。用底物浸泡过夜的AE晶体的X射线衍射分析也证实了3-羧基丙酰基-Ala-Ala-Ala-对硝基苯胺（SAN）与AE活性位点的络合。在差分图中未观察到对硝基苯胺部分。结果表明，AE与火鸡卵类粘蛋白抑制剂的解离常数（Kd）为0.3±0.06μM，而PE为0.10μM。AE-SAN复合物的Kd（0.2 mM）与SAN对PE的米氏常数（1.0 mM）相当。许多抑制剂，如与PE形成半缩酮加合物的弹性蛋白酶抑制剂，而其他如作为活性位点丝氨酸残基酰化剂的β-内酰胺类，与AE的结合亲和力低于与PE的结合亲和力。肽基氯甲烷（乙酰基-Ala-Ala-Pro-Ala-CH2Cl）与AE的结合未显示组氨酸烷基化的证据。苯并异噻唑啉酮和3,4-二氯异香豆素与PE的结合常数与其与AE的结合常数相差不到4.0倍。丝氨酸-195的羟基对这些抑制剂与PE在其非共价复合物中的结合贡献相对较小，尽管它们通过酰化机制使PE失活。这些结果表明，与PE在催化特性中的重要作用相反，PE中丝氨酸-195上的羟基在配体结合的能量学中是次要的。
