Date of Award

1-1-1986

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry

Abstract

The ionization potentials of carboxyl groups in trypsin were studied with spectroscopic and kinetic methods. A mechanism based inhibitor (p-nitrophenol anthranilate) was used to block the Ser-195 (acylated enzyme) residue in the active site with a fluorescent anthranilate tag. Fluorescence quenching studies with iodide and acrylamide suggested the pKa value of the His-57 residue adjacent to the anthranilate tag was between 7 and 8. Fourier transform infrared spectroscopy (FT-IR) analysis of the carboxyl groups symmetrical stretch at 1400 cm('-1) showed that all their pKa values were 4.6 or below in the trypsin anthranilate system. FT-IR studies on native trypsin and trypsin with surface carboxyl groups blocked and an unblocked Ser-195 residue revealed two pKa values of 6.2 and 6.8. The latter values correspond with previously reported pKa values for catalytically active carboxyl groups located in the hydrophobic interior of trypsin. A mechanism is proposed that related the depressed pKa value of the carboxyl group (Asp-102) in the acylated trypsin system to an overall catalytic mechanism; in the acylated trypsin it acts as a proton donor toward the acylated Ser-195 and a proton sink when the Ser-195 is free.Forster energy transfer between the tryptophan residues and the anthranilate tag was used as a structural probe. Trypsin anthranilate was placed in varying amounts of denaturant (urea) and the effect on the fluorescence spectra was noted. Comparison of these spectra to those obtained above pH 8.2 revealed a structural change in the enzyme when placed in solutions above pH 8.2. Implications of a structural change are that the hydrophobic environments of interior carboxyl groups may change at pH values above pH 8.2. Previous analyses of pH rate profiles above 8.2 do not account for any change in solvation for the carboxyl groups.Inhibitor interactions with trypsin and chemically modified trypsin were investigated with kinetic methods. For soybean trypsin inhibitor (STI), using lysine methyl ester (Lys-OMe) substrate the mode of inhibition at pH 6 was noncompetitive for native trypsin and chemically modified trypsin. Kinetic studies with the benzoyl arginine ethyl ester substrate also suggested the mode of inhibition to be noncompetitive at pH 8.0. Inhibition constants were determined with the assumption of a noncompetitive mechanism.

Download

Share

COinS