LOCATING THE CATALYTIC WATER MOLECULE IN SERINE PROTEASES
LOCATING THE CATALYTIC WATER MOLECULE IN SERINE PROTEASES
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DOI：
10.1126/science.8342029
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发表时间：
1993-07-30
期刊：
影响因子：
56.9
通讯作者：
FLETTERICK, RJ
中科院分区：
文献类型：
--
作者：
PERONA, JJ;CRAIK, CS;FLETTERICK, RJ
5.残基41的羰基的位置（胰凝乳蛋白酶编号系统）在胰蛋白酶样丝氨酸蛋白酶的所有已知结构中是保守的[Brookhaven蛋白质数据库（PDB）给出了编码]：胰凝乳蛋白酶（4CHA），胰腺弹性蛋白酶（3EST），大鼠肥大细胞蛋白酶11（3RP 2），嗜中性粒细胞弹性蛋白酶（1HNE），tonin（1 TON），猪胰激肽释放酶A（2 PKA），胰蛋白酶（3 PTN）、α-裂解蛋白酶（1 P01）、灰色葡萄球菌胰蛋白酶（1 SGT）和S.灰色蛋白酶B（3SGB）。所有这些酶的羰基氧原子排列在1.5 A以内，7种哺乳动物酶的羰基氧原子排列在1.0 A以内。连接残基Cys 42至Cys 58的二硫键可能部分地严格保守，因为它是维持位置41处的羰基氧相对于催化His57.6的取向所必需的。枯草杆菌蛋白酶BPN ′ [CS Wright，RA奥尔登，J. Kraut，自然221，235（1969）; PDB 1 SBT]和枯草杆菌蛋白酶Carlsberg与eglin C的复合物[W. Bode，E. Papamoukos，D.穆西尔湾Seemueller，H. Fritz，EMBO J.5，813（1986）; PDB 1CSE]通过使用催化三联体丝氨酸、组氨酸和组氨酸残基的侧链原子叠加到嗜中性粒细胞弹性蛋白酶（HNE）的侧链上。两个枯草杆菌蛋白酶结构中残基Asn 218的羰基氧相对于HNE残基41的等效羰基氧被置换2至3 A。然而，相对于催化三联体残基，该基团的取向和距离在每种酶中是相似的。7. His 57在离去基团质子化过程中是否移位的问题仍然没有解决。在溶液中进行的各种研究已经得出了相互矛盾的结论[WW Bachovchin，Bio-chemistry 25，7751（1986）; J. Kraut，Annu. Rev.Biochem.46，331（1977）; JH Wang，Proc. Nati. Acad. Sci. USA 66，874（1970）]。α-裂解蛋白酶与肽硼酸和膦酸酯过渡态类似物复合物的晶体结构表明，离去基团氧原子（相当于蛋白质底物的P1 ′-氮）与His 57的NE形成氢键[R. Bone，AB Shenvi，C. A. Kettner，D. A. Agard，Biochemistry 26，7609（1987）; R. Bone，N. S. Sampson，PA Bartlett，DA Agard，ibid. 30，2263（1991）]。这表明His 57的小运动可能需要离去基团质子化。
5. The position of the carbonyl group of residue 41 (chymotrypsin numbering system) is conserved in all known structures of trypsin-like serine proteases [Brookhaven Protein Data Bank (PDB) iden-tification codes given]: chymotrypsin (4CHA), pancreatice elastase (3EST), rat mast cell protease 11 (3RP2), neutrophil elastase (1HNE), tonin (1TON), porcine pancreatic kallikrein A (2PKA), trypsin (3PTN), and the bacterial enzymes a-lytic protease (1 P01), Staphlococcus griseus trypsin (1 SGT), and S. griseus proteinase B (3SGB). The carbonyl oxygen atom aligns within 1.5 A for all of these and within 1.0 A for the seven mammalian enzymes. The disulfide bond linking residues Cys42 to Cys58 maybe strictly conserved in part because it is required for maintaining the orientation of the carbonyl oxygen at position 41 relative to the catalytic His57.6. The high-resolution crystal structures of subtilisin BPN'[CS Wright, RA Alden, J. Kraut, Nature 221, 235 (1969); PDB 1SBT] and of subtilisin Carlsberg complexes with eglin C [W. Bode, E. Papamoukos, D. Musil, U. Seemueller, H. Fritz, EMBO J. 5, 813 (1986); PDB 1CSE] were superimposed onto that of the neutrophil elastase (HNE) with the use of side chain atoms of the catalytic triad serine, histidine, and aspartateresidues. The carbonyl oxygen of residue Asn218 in the two subtilisin structures is displaced by 2 to 3 A relative to the equivalent carbonyl oxygen of residue 41 of HNE. However, the orientation and distance of this group with respect to the catalytic triad residues are similar in each enzyme. 7. The issue of whether His57 shifts position during leaving-group protonation is still unresolved. Various studies conducted in solution have reached conflicting conclusions [WW Bachovchin, Bio-chemistry 25, 7751 (1986); J. Kraut, Annu. Rev. Biochem. 46, 331 (1977); JH Wang, Proc. Nati. Acad. Sci. USA 66, 874 (1970)]. Crystal struc-tures of a-lytic protease complexes with peptide boronic acid and phosphonate transition state analogs show that the leaving-group oxygen atom (equivalent to the P1'-nitrogen of a protein sub-strate) is hydrogen-bonded to NEof His57 [R. Bone, AB Shenvi, C. A. Kettner, D. A. Agard, Biochemistry 26, 7609 (1987); R. Bone, N. S. Sampson, PA Bartlett, DA Agard, ibid. 30, 2263 (1991)]. Thissuggests that little movement of His57 may be required for leaving-group protonation.