Interaction of Inactive Derivatives of Chymotrypsin and Trypsin with Protein Inhibitors

From the ¹ From the Department of Food Science and Technology, University of California, Davis, California 95616

The ability of several catalytically inactive derivatives of -chymotrypsin and trypsin to react with naturally occurring protein inhibitors was studied. Two types of inactive derivatives were used: (a) modified seryl derivatives of chymotrypsin, tosyl-chymotrypsin, and anhydro-chymotrypsin prepared by base elimination of the tosyl group; (b) modified histidyl derivatives, the derivative of l-1-tosylamido-2-phenylethyl chloromethyl ketone and chymotrypsin and the derivative of 1-chloro-3-tosylamido-7-amino-2-heptanone and trypsin. The inhibitors were several ovomucoids from different avian species and inhibitors from beans and potato. The formation of the complexes was established by competitive enzymatic assays and by electrophoresis in gel.

Tosyl-chymotrypsin did not form a complex with an inhibitor of chymotrypsin but the anhydro-chymotrypsin did form a complex. The inactive histidyl derivatives of both chymotrypsin and trypsin formed complexes with several inhibitors of chymotrypsin and trypsin, respectively. The rate of interaction of anhydro-chymotrypsin with inhibitors was similar to that of native chymotrypsin, while the rates of the histidyl derivatives of chymotrypsin and trypsin were slower.

The naturally occurring protein inhibitors were found to serve as useful tools for studying the effects of chemical modifications on the tertiary structure of proteolytic enzymes. Catalytic activity of the enzymes was not essential for the formation of complexes. As evidenced by the formation of complexes, specific substitutions of the catalytic histidyl residues of both chymotrypsin and trypsin do not greatly affect their tertiary structures. Substitution on the catalytic seryl residue of chymotrypsin changes the tertiary structure or causes steric effects, while modification of the seryl residues by base elimination of the tosyl group does not result in any gross structural changes.

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