1. Reinvestigation of the phenacyl bromide modification of α-chymotrypsin
- Author
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George I. Glover, John R. Petersen, and Patrick S. Mariano
- Subjects
Alkylation ,Sulfonium ,Sulfonium Compounds ,Salt (chemistry) ,Hydroxylamines ,Phenacyl ,Biochemistry ,Medicinal chemistry ,Structure-Activity Relationship ,chemistry.chemical_compound ,Methionine ,Bromide ,Animals ,Chymotrypsin ,Mercaptoethanol ,chemistry.chemical_classification ,Binding Sites ,biology ,Phenacyl bromide ,Acetophenones ,Sulfoxide ,Hydrogen-Ion Concentration ,chemistry ,Sulfoxides ,biology.protein ,Cattle - Abstract
The modification of alpha-chymotrysin with phenacyl bromide has been reinvestigated over a wide pH range. Evidence is presented that indicates that the nature of the phenacyl-modified enzymes prepared by this reaction is dependent upon the pH of the reaction medium. The phenacyl alpha-chymotrypsin produced at low pH is most probably the Met-192 phenacylsulfonium salt, as proposed earlier, since it readily undergoes dealkylation using 2-mercaptoethanol. However, the phenacyl-enzyme prepared at neutral pH possesses a much reduced enzymatic activity and does not react with 2-mercaptoethanol to regenerate native alpha-chymotrypsin. In addition, incubation of the Met-192 phenacyl sulfonium enzyme at neutral pH causes a smooth irreversible change to the new phenacyl-enzyme as monitored by changes in enzymatic activity, susceptibility to dealkylation using 2-mercaptoethanol, and ultraviolet difference absorption spectral properties. The stoichiometries of both the low and neutral pH modification reactions have been determined, using [carbonyl-14C]phyenacyl bromide, to be 1 phenacyl group/enzyme molecule. In efforts to obtain information about the nature and mechanism of formation of the phenacyl alpha-chymotrypsin produced at neutral pH, alkylation reactions of modified alpha-chymotrypsins produced by His-57 functionalization with tosylphenylalanine chloromethyl ketone and by Met-192 oxidation to the sulfoxide have been investigated. The combined results of these studies have been initially interpreted in terms of a neutral pH, phenacyl bromide modification resulting in formation of a new modified enzyme via the Met-192 sulfonium salt.
- Published
- 1976
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