IDENTIFICATION OF THE ACTIVE-SITE NUCLEOPHILE IN 6-PHOSPHO-BETA-GALACTOSIDASE FROM STAPHYLOCOCCUS-AUREUS BY LABELING WITH SYNTHETIC INHIBITORS

Kinetic parameters for the inactivation of the 6-phospho-beta-galactos idase of Staphylococcus aureus by a series (fluoro, chloro, bromo) of phenyl-2-deoxy-2-halogeno-galactoside-6-phosphates have been determine d. These inhibitors function by the formation of a stabilised glycosyl -enzyme intermediate. Inactivation and reactivation studies indicate t hat the fluoro derivative is formed most rapidly, but is also hydrolys ed fastest. The chloro derivative forms the most stable covalent inter mediate. HPLC profiles of V8-protease digestion of native and inhibite d protein show significant differences, whereas the inhibited 6-phosph o-beta-galactosidase and a point mutant of 6-phospho-beta-galactosidas e (E375Q) yield the same proteolytic fragments. The suggestion that E3 75 is derivatised is strengthened by matrix-assisted laser-desorption ionisation mass spectrometry experiments which show that the two pepti des, residues 336-375 and 376-383, are not produced, due to the absenc e of the expected cleavage at residues 375 and 376. The reason for the altered proteolysis pattern of the inhibited protein is blocking of t he respective V8 cleavage site due to the chemical reaction of the inh ibitor at position 375. Specific modification of the glycosyl bond bet ween 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 w as isolated by Edman degradation of trypsin-digested V8-peptide. It wa s shown to be identical to an authentic, synthetic sample. From this, it is evident that E375 is the active-site nucleophile of 6-phospho-ga lactosidase, consistent with previous findings for enzymes in this fam ily.