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The three-dimensional crystal structure of bovine trypsinogen at approximately
pH 7.5 was initially solved at 2.6 A resolution using the multiple isomorphous
replacement method. Preliminary refinement cycles of the atomic coordinates
trypsinogen have been carried out first to a resolution of 2.1 A, and later to
1.9 A, using constrained difference Fourier refinement; During the process,
structure factors Fc and phi c were calculated from the trypsinogen structure
and final interpretation was based on an electron-density map computed with
terms (2 Fo - Fc) and phases phic at a resolution of 1.9 A. Crystals of
trypsinogen grown from ethanol-water mixtures are trigonal with space group
P3121, and cell dimension a = 55.17 A and c = 109.25 A. The structure is
compared with the bovine diisopropylphosphoryltrypsin structure at approximately
pH 7.2, oirginally determined from orthohombic crystals by Stroud et al.
(Stroud, R.M., Kay L.M., and Dickerson, R.E. (1971), Cold Spring Harbor Symp.
Quant. Biol. 36, 125-140; Stroud, R.M., Kay, L.M., and Dickerson, R.E. (1974),
J. Mol. Biol. 83, 185-208), and later refined at 1.5 A resolution by Chambers
and Stroud (Chambers, J.L., and Stroud, R.M. (1976), Acta Crystallogr. (in
press)). At lower pH, 4.0-5.5 diogen, with cell dimensions a = 55.05 A and c =
109.45 A. This finding was used in the solution of the six trypsinogen
heavy-atom derivatives prior to isomorphous phase analysis, and as a further
basis of comparison between trypsinogen and the low pH trypsin structure. There
are small differences between the two diisopropylphosphoryltrypsin structures.
Bovine trypsinogen has a large and accessible cavity at the site where the
native enzyme binds specific side chains of a substrate. The conformation and
stability of the binding site differ from that found in trypsin at approximately
pH 7.5, and from that in the low pH form of diisopropylphosphoryltrypsin. The
catalytic site containing Asp-102, His-57, and Ser-195 is similar to that found
in trypsin and contains a similar hydrogen-bounded network. The carboxyl group
of Asp-194, which is salt bridged to the amino terminal of Ile-16 in native
trypsin or other serine proteases, is apparently hydrogen bonded to internal
solvent molecules in a loosely organized part of the zymogen structure. The
unusually charged N-terminal hexapeptide of trypsinogen, whose removal leads to
activation of the zymogen, lies on the outside surface of the molecule. There
are significant structural changes which accompany activation in neighboring
regions, which include residues 142-152, 215-550, 188A-195. The NH group of
Gly-193, normally involved in stabilization of reaction intermediates (Steitz,
T.A., Henderson, R., and Blow, D.M. (1969), J. Mol. Biol. 46, 337-348;
Henderson, R. (1970), J. Mol. Biol. 54, 341-354; robertus, J.D., Kraut, J.,
Alden, R.A., and Birkoft, J.J. (1972), Biochemistry 11, 4293-4303) in the
enzyme, is moved 1.9 A away from its position in trypsin...
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