On protein data bank entries, bovine beta-trypsin, and the mechanism of action of calcium in trypsin autolysis.
Item
-
Title
-
On protein data bank entries, bovine beta-trypsin, and the mechanism of action of calcium in trypsin autolysis.
-
Identifier
-
AAI9207053
-
identifier
-
9207053
-
Creator
-
Buono, Ronald Alexander.
-
Contributor
-
Adviser: Michael N. Liebman
-
Date
-
1991
-
Language
-
English
-
Publisher
-
City University of New York.
-
Subject
-
Chemistry, Biochemistry | Biology, Animal Physiology
-
Abstract
-
A statistical analysis of comparisons among several types of crystalline proteins was conducted to construct a library of conformational changes in proteins with identical primary sequences. Specific structural and thermal parameter analyses indicated that bovine beta-trypsin is a typical globular protein, with characteristics similar to most of the other structures deposited in the Protein Data Bank. Its active site is particularly well-conserved, with the calcium-binding and autolysis sites exhibiting progressively greater conformational and thermal variabilities.;Molecular modeling of the beta-trypsin autolysis dimer showed that the P1{dollar}\sp\prime{dollar}-P5{dollar}\sp\prime{dollar} residues of the 'substrate' are responsible for the majority of the steric overlap in the dimer. Molecular mechanics minimizations indicated that backbone atoms must adapt significantly at the protein-protein interface in order to produce the Michaelis complex. The extent of this adaptation indicates that these changes are the result of an activated process.;Fourier-transform infrared spectroscopic experiments showed that no detectable conformational changes occur to the secondary structure of two inhibited forms of bovine beta-trypsin upon calcium binding. Furthermore, the appearance of a band at 1699 cm{dollar}\sp{lcub}-1{rcub}{dollar} in benzamidine-inhibited trypsin is consistent with the observed asymmetric crystal structure interaction between the amidinium group of the inhibitor and the carbonyl group of 189D in the enzyme active site.;Analyses of the solvent accessibilities, thermal parameters, and steric overlap resulting from dimerization at the 16 basic sites of trypsin led to the conclusion that the 145K site is the preferred initial substrate in autolysis if mobility of the P1{dollar}\sp\prime{dollar}-P5{dollar}\sp\prime{dollar} residues is important for allowing bulk water to enter for the deacylation step of proteolysis. A conformational change at the alpha site, after cleavage of beta-trypsin, is also inferred from these analyses.;It has been proposed by others that calcium slows trypsin autolysis by the induction of a "conformational change" in the autolysis loop of the 'substrate'. However, the combination of molecular modeling, crystal structure and thermal parameter analyses, FTIR spectroscopic evidence, and consideration of other experiments performed on both trypsin and chymotrypsin, suggest that calcium's mechanism of action is of a specific electrostatic origin.
-
Type
-
dissertation
-
Source
-
PQT Legacy CUNY.xlsx
-
degree
-
Ph.D.
