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Evidence for a pH-dependent irreversible formation of a stable conformation of phenacyl-alpha-chymotrypsin
Abstract:
A reinvestigation of the modification reactions of alpha-chymotrypsin with phenacyl bromide was carried out. Results conclusively demonstrate that the chemically and physically different modified enzymes prepared at pH 4 and at pH 7 both contain the phenacyl group at methionine-192 in the sulphonium salt form. Evidence to suppoort this conclusion derives from 13C nuclear-magnetic-resonance spectroscopic observations on [methylene-13C]phenacyl-enriched enzymes. More conclusively, the methionine-192-containing C-chain, derived by performic acid oxidative cleavage of radioactively-labelled enzyme prepared at pH 7, was shown to contain the phenacyl moiety and to undergo dealkylation by 2-mercaptoethanol with loss of this moiety. In addition, thermolytic cleavage of the high-pH enzyme results in fragmentation of the polypeptide chain in a fashion analogous to model reactions of phenacylmethionyl dipeptides and other methionine-192 sulphonium salts. A rationalization of the unusual nature of the high-pH phenacyl-modified enzyme based on the irreversible formation of stable conformation in which the phenacyl moiety is rigidly located in interior regions of the enzyme is presented and discussed.
Insights
This study reveals that phenacyl bromide modifies alpha-chymotrypsin at methionine-192, forming a sulphonium salt. This modification occurs regardless of pH, influencing enzyme conformation and stability.
Area of Science:
- Biochemistry
- Enzymology
- Chemical Modification
Background:
- Alpha-chymotrypsin is a key digestive enzyme.
- Understanding enzyme modification is crucial for protein chemistry.
Purpose of the Study:
- To reinvestigate the modification reactions of alpha-chymotrypsin with phenacyl bromide.
- To elucidate the specific site and form of phenacyl modification.
- To understand the structural and conformational consequences of this modification.
Main Methods:
- 13C nuclear-magnetic-resonance spectroscopy on enriched enzymes.
- Performic acid oxidative cleavage of radioactively-labelled enzyme.
- Thermolytic cleavage of modified enzyme.
- Comparison with model reactions of phenacylmethionyl dipeptides.
Main Results:
- Phenacyl group is located at methionine-192 in a sulphonium salt form at both pH 4 and pH 7.
- Radioactive labelling and dealkylation experiments confirm phenacyl moiety attachment and release.
- High-pH modification leads to stable conformations with rigidly located phenacyl groups.
Conclusions:
- The modification of alpha-chymotrypsin by phenacyl bromide consistently targets methionine-192 as a sulphonium salt.
- The reaction conditions (pH) influence the enzyme's conformational stability post-modification.
- The findings provide insights into enzyme structure-function relationships and chemical modification mechanisms.