Helland Ronny, Czapinska Honorata, Leiros Ingar, Olufsen Magne, Otlewski Jacek, Smalås Arne O
Norwegian Structural Biology Centre, Faculty of Science, University of Tromsø, 9037 Tromsø, Norway.
J Mol Biol. 2003 Oct 31;333(4):845-61. doi: 10.1016/j.jmb.2003.08.059.
Crystal structures of P1 Gly, Val, Leu and Phe bovine pancreatic trypsin inhibitor (BPTI) variants in complex with two serine proteinases, bovine trypsin and chymotrypsin, have been determined. The association constants for the four mutants with the two enzymes show that the enlargement of the volume of the P1 residue is accompanied by an increase of the binding energy, which is more pronounced for bovine chymotrypsin. Since the conformation of the P1 side-chains in the two S1 pockets is very similar, we suggest that the difference in DeltaG values between the enzymes must arise from the more polar environment of the S1 site of trypsin. This results mainly from the substitutions of Met192 and Ser189 observed in chymotrypsin with Gln192 and Asp189 present in trypsin. The more polar interior of the S1 site of trypsin is reflected by a much higher order of the solvent network in the empty pocket of the enzyme, as is observed in the complexes of the two enzymes with the P1 Gly BPTI variant. The more optimal binding of the large hydrophobic P1 residues by chymotrypsin is also reflected by shrinkage of the S1 pocket upon the accommodation of the cognate residues of this enzyme. Conversely, the S1 pocket of trypsin expands upon binding of such side-chains, possibly to avoid interaction with the polar residues of the walls. Further differentiation between the two enzymes is achieved by small differences in the shape of the S1 sites, resulting in an unequal steric hindrance of some of the side-chains, as observed for the gamma-branched P1 Leu variant of BPTI, which is much more favored by bovine chymotrypsin than trypsin. Analysis of the discrimination of beta-branched residues by trypsin and chymotrypsin is based on the complexes with the P1 Val BPTI variant. Steric repulsion of the P1 Val residue by the walls of the S1 pocket of both enzymes prevents the P1 Val side-chain from adopting the most optimal chi1 value.
已确定P1位点为甘氨酸、缬氨酸、亮氨酸和苯丙氨酸的牛胰蛋白酶抑制剂（BPTI）变体与两种丝氨酸蛋白酶——牛胰蛋白酶和胰凝乳蛋白酶形成复合物的晶体结构。这四种突变体与这两种酶的缔合常数表明，P1残基体积的增大伴随着结合能的增加，这在牛胰凝乳蛋白酶中更为明显。由于两种酶S1口袋中P1侧链的构象非常相似，我们认为酶之间ΔG值的差异一定源于胰蛋白酶S1位点更具极性的环境。这主要是由于在胰凝乳蛋白酶中观察到的Met192和Ser189被胰蛋白酶中的Gln192和Asp189取代。胰蛋白酶S1位点更具极性的内部由该酶空口袋中溶剂网络的更高有序性反映出来，正如在这两种酶与P1甘氨酸BPTI变体的复合物中所观察到的那样。胰凝乳蛋白酶对大的疏水性P1残基的更优结合也通过该酶同源残基容纳时S1口袋的收缩反映出来。相反，胰蛋白酶的S1口袋在结合此类侧链时会扩张，可能是为了避免与口袋壁的极性残基相互作用。两种酶之间的进一步区分是通过S1位点形状的微小差异实现的，这导致了一些侧链的空间位阻不均等，如在BPTI的γ-分支P1亮氨酸变体中观察到的那样，牛胰凝乳蛋白酶比胰蛋白酶更倾向于结合该变体。对胰蛋白酶和胰凝乳蛋白酶对β-分支残基的区分分析是基于与P1缬氨酸BPTI变体的复合物。两种酶S1口袋壁对P1缬氨酸残基的空间排斥阻止了P1缬氨酸侧链采用最优化的χ1值。
