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Identification of the active-site nucleophile in 6-phospho-beta-galactosidase from Staphylococcus aureus by labelling
P Staedtler1, S Hoenig, R Frank
1Arbeitsgruppe Physiologie der Mikroorganismen, Abteilung Biologie, Ruhr-Universität Bochum, Germany.
Abstract:
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.
Insights
This study identifies glutamic acid at position 375 (E375) as the active-site nucleophile in Staphylococcus aureus 6-phospho-beta-galactosidase. Halogenated inhibitors reveal E375
Area of Science:
- Biochemistry
- Enzyme kinetics
- Protein chemistry
Background:
- Staphylococcus aureus 6-phospho-beta-galactosidase is a key enzyme in bacterial metabolism.
- Understanding its active site is crucial for developing targeted inhibitors.
Purpose of the Study:
- To determine the kinetic parameters for the inactivation of Staphylococcus aureus 6-phospho-beta-galactosidase using halogenated dinitrophenyl galactoside phosphates.
- To identify the specific amino acid residue at the active site responsible for inhibitor interaction.
Main Methods:
- Enzyme kinetics studies to determine inactivation and reactivation rates.
- High-performance liquid chromatography (HPLC) to analyze V8-protease digestion patterns.
- Matrix-assisted laser-desorption ionization mass spectrometry (MALDI-MS) to identify peptide modifications.
- Edman degradation to confirm the identity of the modified residue.
Main Results:
- Halogenated dinitrophenyl galactoside phosphates act as inhibitors by forming a stabilized glycosyl-enzyme intermediate.
- The fluoro derivative reacts fastest but is least stable; the chloro derivative forms the most stable intermediate.
- Proteolytic digestion and mass spectrometry revealed that residue E375 is derivatized by the inhibitor, blocking V8 cleavage.
- Aminolysis and Edman degradation confirmed E375 as the active-site nucleophile.
Conclusions:
- Glutamic acid at position 375 (E375) is the active-site nucleophile of Staphylococcus aureus 6-phospho-beta-galactosidase.
- The findings are consistent with the known mechanism of other enzymes in this family.
- This identification provides a basis for designing more specific enzyme inhibitors.