Preferential nitration with tetranitromethane of a specific tyrosine residue in penicillinase from Staphylococcus aureus PCl. Evidence that the preferentially nitrated residue is not part of the active site but that loss of activity is due to intermolecular cross-linking

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Abstract
Nitration of tyrosine residues of staphylococcal penicillinase [EC 3.5.2.6] was accompanied by a partial loss of enzymic activity, which was not readily explained by nitration of a single residue. Loss of activity correlated with low recovery of tyrosine plus nitrotyrosine, which was consistent with cross-linking. The fraction of treated enzyme that was eluted from Sephadex G-75 earlier than native penicillinase was similar to the fraction of enzyme activity lost. Protein eluted in positions corresponding to monomer, dimer and higher oligomers, respectively, showed major bands in corresponding positions in sodium dodecyl sulfate/polyacrylamide-gel electrophoresis, indicating that the increase in MW was due to intermolecular cross-linking. Monomeric enzyme containing up to 4 mol of nitrotyrosine/mol retained full catalytic activity. Dimeric enzyme retained 50% of normal activity, but higher oligomers retained an average of 8-15% of normal activity. Monomeric enzyme isolated after treatment with equimolar tetranitromethane was nitrated predominantly at tyrosine-72. Reaction of reduced nitrated monomer with 1,5-difluoro-2,4-dinitrobenzene gave a monomeric, apparently cross-linked product will full catalytic activity. Tyrosine-72 apparently plays no part in the active site. Its preferential nitration may be due to its being insufficiently exposed to be available for intermolecular cross-linking. This property may make it useful for attachment of a reporter group.