Active-site labeling of an aminoglycoside antibiotic phosphotransferase (APH(3')-IIIa).
The aminoglycoside antibiotics are inactivated by modifying enzymes that are now widely distributed in many pathogenic bacteria. This situation threatens the continued use of these clinically important drugs. We have undertaken studies to understand the molecular mechanism of aminoglycoside resistance, and we report the affinity labeling of the enterococcal aminoglycoside 3'-phosphotransferase, APH(3')-IIIa, with an electrophilic ATP analogue, 5'-[p-(fluorosulfonyl)benzoyl]adenosine (FSBA). Incubation of purified APH(3')-IIIa with FSBA resulted in time-dependent irreversible inactivation of enzyme activity with a binding constant, Ki, of 0.406 mM and a rate of maximal inactivation, kmax, of 0.086 min-1. Addition of ATP completely protected the enzyme from inactivation, consistent with labeling of the ATP binding site. Reaction of APH(3')-IIIa with [14C]FSBA showed that inactivated APH(3')-IIIa incorporates 1 mol of FSBA/mol of enzyme. Peptide mapping of FSBA-inactivated APH(3')-IIIa resulted in the identification of two peptide peaks with highly increased absorbance at 260 nm, indicative of covalent labeling with FSBA. Analysis by electrospray ionization mass spectrometry 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 region of APHs in ATP binding, a region of these enzymes which has heretofore not been considered for involvement in substrate binding.
McKay
G A
GA
Department of Biochemistry, McMaster University, Hamilton, Ontario, Canada.
Robinson
R A
RA
Lane
W S
WS
Wright
G D
GD
D000241:Adenosine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D000345:Affinity Labels; D000595:Amino Acid Sequence; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000201:enzymology; D019868:Kanamycin Kinase; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D010449:Peptide Mapping; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism
D000345:Affinity Labels; D010446:Peptide Fragments; C010959:5'-(4-fluorosulfonylbenzoyl)adenosine; D000255:Adenosine Triphosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); D019868:Kanamycin Kinase; D014357:Trypsin; D000241:Adenosine