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Published on: September 30, 2011
Protease II from Escherichia coli. Purification and characterization
The Journal of Biological Chemistry
|October 10, 1975
Summary
Researchers purified protease II, an endopeptidase from Escherichia coli, revealing its distinct properties and substrate specificity. This enzyme degrades specific proteins, highlighting the role of substrate conformation in proteolysis.
Area of Science:
- Biochemistry
- Enzymology
- Microbial Physiology
Background:
- Escherichia coli possesses multiple endopeptidases.
- Previous work identified two types of endopeptidases.
- Protease I, a chymotrypsin-like enzyme, was previously isolated.
Purpose of the Study:
- To purify and characterize a second endopeptidase from Escherichia coli, designated protease II.
- To investigate the enzymatic properties and substrate specificity of protease II.
- To compare protease II with other known proteases, including protease I and trypsin.
Main Methods:
- Purification of protease II using a multi-step procedure.
- Homogeneity assessment via electrophoresis and gel filtration.
- Molecular weight determination using three different methods.
- Enzyme kinetics studies (Michaelis constant, inhibition assays).
- Substrate degradation assays using axocasein, beta-galactosidase, and aspartokinase I/III.
Main Results:
- Protease II was purified 13,500-fold with 24% recovery, appearing homogeneous with a molecular weight of approximately 58,000.
- Optimal pH is 8; activity is stimulated by calcium ions but unaffected by chelating or sulfhydryl reagents.
- Enzyme exhibits esterase activity on lysine and arginine esters, inhibited by diisopropylfluorophosphate and tosyl lysine chloromethyl ketone, suggesting active site serine and histidine.
- Protease II is distinct from protease I and trypsin, showing low activity on axocasein, no effect on native beta-galactosidase, but readily degrading aspartokinase I and III, which are resistant when allosterically regulated.
Conclusions:
- Protease II is a novel serine/histidine-containing endopeptidase from Escherichia coli with unique substrate specificity.
- Substrate conformation significantly influences protease susceptibility, as demonstrated by the differential degradation of aspartokinase.
- These findings contribute to understanding the regulation of protein degradation in vivo through conformational changes.
