Inhibited Bovine Trypsin, and Bovine Trypsinogen : the Refined Structure and Mechanism of a Not-Too-Serene Protease

Author: Chambers, John L.

Year: 1977

Degree: Dissertation (Ph.D.)

Advisor: Stroud, Robert M.

Committee Member: Unknown, Unknown

Option: Chemistry

DOI: 10.7907/69pr-0q16

Abstract

Refined three-dimensional structures of diisopropylphosphoryl (DIP)-inhibited bovine trypsin and of bovine trypsinogen are described, as determined by x-ray crystallography. A constrained difference Fourier method for rapid, economical protein structure refinement was developed and applied to these structures. The refinement system, which has been written to operate on a minicomputer, has reduced the standard crystallographic R-factor for DIP-trypsin from 47.2% at 2.7 Å resolution to 21.5% at 1.5 Å resolution, making it one of the most highly and inexpensively refined protein structures to date. For trypsinogen, R is 31% at 1.9 Å resolution, and refinement of both structures is nearing completion. The refined structures, the mechanism of serine proteases, and the mechanism of activation of their zymogens are discussed.

During data collection for the studies described, new, more efficient techniques of correction for background radiation in protein data measured by x-ray diffractometry were developed.

Also presented are the structures of trypsin inhibited by the metal ions Ag+ and Cu2+. These ions, which are powerful trypsin and chymotrypsin inhibitors, bind tightly and specifically at the active site of the enzyme, between Asp 102 Oδ1 and His 57 Nδ1. They may thus be used as specific probes of the active site of serine proteases.

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