ACTIVE-SITE LABELING OF AN AMINOGLYCOSIDE ANTIBIOTIC PHOSPHOTRANSFERASE (APH(3')-IIIA)

The aminoglycoside antibiotics are inactivated by modifying enzymes th at are now widely distributed in many pathogenic bacteria. This situat ion threatens the continued use of these clinically important drugs. W e have undertaken studies to understand the molecular mechanism of ami noglycoside resistance, and we report the affinity labeling of the ent erococcal aminoglycoside 3'-phosphotransferase, APH(3')-IIIa, with an electrophilic ATP analogue, 5'-[p-(fluorosulfonyl)benzoyl]adenosine (F SBA). Incubation of purified APH(3')-IIIa with FSBA resulted in time-d ependent irreversible inactivation of enzyme activity with a binding c onstant, K-i, of 0.406 mM and a rate of maximal inactivation, k(max), of 0.086 min(-1) Addition of ATP completely protected the enzyme from inactivation, consistent with labeling of the ATP binding site. Reacti on of APH(3')-IIIa with [C-14]FSBA showed that inactivated APH(3')-III a incorporates 1 mol of FSBA/mol of enzyme. Peptide mapping of FSBA-in activated APH(3')-IIIa resulted in the identification of two peptide p eaks with highly increased absorbance at 260 nm, indicative of covalen t labeling with FSBA. Analysis by electrospray ionization mass spectro metry and Edman degradation revealed two tryptic peptides, Val31-Lys44 and Leu34-Arg49, which incorporated the FSBA label at Lys33 and Lys44 , respectively. This establishes the importance of the N-terminal regi on of APHs in ATP binding, a region of these enzymes which has heretof ore not been considered for involvement in substrate binding.