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Serine Proteases: Structure, Mechanism, and Role in Eucaryotic Cell Transformation

Citation

Krieger, Monty (1976) Serine Proteases: Structure, Mechanism, and Role in Eucaryotic Cell Transformation. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/h6dh-m388. https://resolver.caltech.edu/CaltechTHESIS:01292025-223653680

Abstract

The structure and catalytic mechanism of the serine protease trypsin was investigated using x-ray crystallography, enzyme kinetics and hydrogen isotope exchange. The role of serine proteases in eucaryotic cell transformation was examined by studying the binding of 125I-labeled dog plasminogen to normal and SV40-transformed Balb/c 3T3 cells.

X-ray structural analysis using the difference Fourier technique showed that benzamidine, a non-covalent, reversible inhibitor of trypsin, binds in the specificity binding pocket of the enzyme and blocks substrate access to the active center. Benzamidine binding provided a reasonable model for the binding of specific side chains and permitted us to construct a model for specific substrate binding. The difference Fourier technique was also used to show that silver ions inhibit trypsin by binding between the aspartic acid and histidine side chains in the catalytic site. This observation was used to assign difference infrared spectral peaks to the side chain of Asp 102 in the catalytic site. A new level in understanding the catalytic mechanism was achieved using this information.

In the course of these crystallographic investigations, new, more efficient and accurate techniques of data collection were developed based upon a detailed study of the sources of background radiation in x-ray diffractometry. These techniques involve new methods for reducing and accurately accounting for background effects.

A pH-dependent conformational change could introduce artifacts in the determination of the pKa's of catalytic groups using either rapid kinetic or slower spectroscopic techniques. The rate of Nα-carbobenzoxy-L-lysine-p-nitrophenyl ester (CLNE) hydrolysis by trypsin over the range pH 2 to pH 5 was observed to test for such a conformational change. Pre-incubating the enzyme at pH 2 and pH 6.9 for up to three hours had no effect on the hydrolysis rate; therefore, within these limits, there was no pH-dependent conformational change which directly affected catalysis.

In an attempt to determine the pKa of His 57 directly, the exchange of tritum with the C-2 protons in the histidine side chains of trypsin was measured. This exchange was found to be slower than any previously measured C-2 tritium exchange. The half-time for exchange into His 57 was 73 days, and this apparently took place when the active site imidazole had rotated away from its active conformation. The apparent pKa of His 57 in this inactive "out" conformation was 6.6.

Increased proteolytic activity exhibited by many cells after transformation is due to plasminogen activation to the trypsin-like enzyme plasmin. Plasmin is responsible for a number of morphological changes which accompany transformation and some experiments have suggested that plasmin or plasminogen might bind to transformed cell surfaces in preference to normal cell surfaces. We therefore studied the time dependence of plasminogen binding to 3T3 and SV 3T3 cells. The binding to 3T3 cells on a per-cell basis either decreased during the course of a three-day incubation or decreased between the first and second days and then rose again on the third day ("V" shaped binding curve). The binding to the SV 3T3 cells was usually "V" shaped. After three days of incubation, all of the plasminogen bound to the SV 3T3 cells had been degraded while there was still substantial plasminogen associated with the 3T3 cells. The degradation by the transformed cells was independent of the plasmin-dependent morphological changes normally exhibited by these cells. The uptake of serum plasminogen by the cells apparently involved several processes and may be accompanied by cell-surface proteolysis in the case of the transformed cells.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:(Chemistry)
Degree Grantor:California Institute of Technology
Division:Chemistry and Chemical Engineering
Major Option:Chemistry
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Stroud, Robert M.
Thesis Committee:
  • Unknown, Unknown
Defense Date:26 May 1976
Record Number:CaltechTHESIS:01292025-223653680
Persistent URL:https://resolver.caltech.edu/CaltechTHESIS:01292025-223653680
DOI:10.7907/h6dh-m388
ORCID:
AuthorORCID
Krieger, Monty0000-0003-4541-5181
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:16971
Collection:CaltechTHESIS
Deposited By: Benjamin Perez
Deposited On:30 Jan 2025 17:55
Last Modified:30 Jan 2025 17:56

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