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Eur J Biochem. 1995 Sep 1;232(2):658-63.

Identification of the active-site nucleophile in 6-phospho-beta-galactosidase from Staphylococcus aureus by labelling with synthetic inhibitors.

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Arbeitsgruppe Physiologie der Mikroorganismen, Abteilung Biologie, Ruhr-Universit├Ąt Bochum, Germany.


Kinetic parameters for the inactivation of the 6-phospho-beta-galactosidase of Staphylococcus aureus by a series (fluoro, chloro, bromo) of 2,4-dinitrophenyl-2-deoxy-2-halogeno- galactoside-6-phosphates have been determined. These inhibitors function by the formation of a stabilised glycosyl-enzyme intermediate. Inactivation and reactivation studies indicate that the fluoro derivative is formed most rapidly, but is also hydrolysed fastest. The chloro derivative forms the most stable covalent intermediate. HPLC profiles of V8-protease digestion of native and inhibited protein show significant differences, whereas the inhibited 6-phospho-beta-galactosidase and a point mutant of 6-phospho-beta- galactosidase (E375Q) yield the same proteolytic fragments. The suggestion that E375 is derivatised is strengthened by matrix-assisted laser-desorption ionisation mass spectrometry experiments which show that the two peptides, residues 336-375 and 376-383, are not produced, due to the absence of the expected cleavage at residues 375 and 376. The reason for the altered proteolysis pattern of the inhibited protein is blocking of the respective V8 cleavage site due to the chemical reaction of the inhibitor at position 375. Specific modification of the glycosyl bond between the inhibitor and E375 by aminolysis with benzylamine generated a glutamatic-acid-5-benzylamide complex at that position in the peptide. The Edman derivative of the modified E375 appears to be stable and was isolated by Edman degradation of trypsin-digested V8-peptide. It was shown to be identical to an authentic, synthetic sample. From this, it is evident that E375 is the active-site nucleophile of 6-phospho-galactosidase, consistent with previous findings for enzymes in this family.

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