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Bovine chymotrypsinogen A

1985; Elsevier BV; Volume: 185; Issue: 3 Linguagem: Inglês

10.1016/0022-2836(85)90074-9

1089-8638

Da‐Cheng Wang, Wolfram Bode, Robert Huber,

Viral Infectious Diseases and Gene Expression in Insects

The X-ray structure of a new crystal form of chymotrypsinogen A grown from ethanol/ water has been determined at 1.8 Å resolution using Patterson search techniques. The crystals are of orthorhombic space group P212121 and contain two molecules in the asymmetric unit. Both independent molecules (referred to as A and B) have been crystallographically refined to a final R value of 0.173 with reflection data to 1.8 Å resolution. Owing to different crystal contacts, both independent molecules show at various sites conformational differences, especially in segments 33–38, 142–153 and 215–222. If these three loops are omitted in a comparison, the root-mean-square (r.m.s.) deviation of the main-chain atoms of molecules A and B is 0.32 Å. If segments 70–79, 143–152 and 215–221 are omitted, a comparison of either molecule A or molecule B with the chymotrypsinogen model of Freer et al. (1970) reveals an r.m.s. deviation of the α-carbon atoms of about 0.7 Å. Compared with the active enzyme, four spatially adjacent peptide segments, in particular, are differently organized in the zymogen: the amino-terminal segment 11–19 runs in a rigid but strained conformation along the molecular surface due to the covalent linkage through Cys1; also segment 184–194 is in a rigid unique conformation due to several mutually stabilizing interactions with the amino-terminal segment; segment 216–222. which also lines the specificity pocket, adapts to different crystal contacts and exists in both chymotrypsinogen molecules in different, but defined conformations; in particular, disulfide bridge 191–220, which covalently links both latter segments, has opposite handedness in molecules A and B; finally, the autolysis loop 142 to 153 is organized in a variety of ways and in its terminal part is completely disordered. Thus, the allosteric activation domain (Huber & Bode, 1978) is organized in defined although different conformations in chymotrypsinogen molecules A and B, in contrast to trypsinogen, where all four homologous segments of the activation domain are disordered. This reflects the structural variability and deformability of the activation domain in serine proteinase proenzymes. If the aforementioned peptide segments are omitted, a comparison of our chymotrypsinogen models with γ-chymotrypsin (Cohen et al., 1981) yields an r.m.s. deviation for α-carbon atoms of about 0.5 Å. The residues of the "active site triad" are arranged similarly, but the oxyanion hole is lacking in chymotrypsinogen. No significant, relative shift of both constituting domains, as described by Birktoft et al. (1976) to occur upon activation, is observed. In agreement with Freer et al. (1970) we conclude that the inadequate formation of the specificity pocket and the absence of the oxyanion hole are the main reasons for the extremely low catalytic power of chymotrypsinogen. Model building experiments indicate that after some slight rearrangements the chymotrypsinogen segment containing the activation cleavage site should fit to the trypsin binding site.