KINETIC MECHANISM OF AMINOGLYCOSIDE PHOSPHOTRANSFERASE TYPE IIIA - EVIDENCE FOR A THEORELL-CHANCE MECHANISM

Bacterial resistance to aminoglycoside-aminocyclitol antibiotics is me diated primarily by covalent modification of the drugs by a variety of enzymes. One such modifying enzyme, the 3'-aminoglycoside phosphotran sferase, which is produced by Gram-positive cocci such as Enterococcus and Streptococcus inactivates a broad range of aminoglycosides by ATP -dependent phosphorylation of specific hydroxyl residues on the antibi otics. Through the use of dead-end and product inhibitor studies, we p resent the first detailed examination of the kinetic mechanism for the 3'-aminoglycoside phosphotransferase-IIIa, Initial velocity patterns deduced from steady-state kinetics indicate a sequential mechanism wit h ordered binding of ATP first followed by aminoglycoside. Dead-end in hibition by AMP and adenylyl-imidodiphosphate is competitive versus AT P and noncompetitive versus kanamycin A. Dead-end inhibition by tobram ycin, a kanamycin analogue lacking a 3'-OH, is competitive versus both kanamycin A and uncompetitive versus ATP, indicative of ordered subst rate binding where ATP must add prior to aminoglycoside addition, Prod uct inhibition by kanamycin phosphate is noncompetitive versus ATP whe n kanamycin A is held at subsaturating concentrations (K-m(kanA)), whe reas no inhibition is observed when the concentration of kanamycin A i s held at 10 K-m(kanA). This is consistent with kanamycin phosphate be ing the first product released followed by ADP release. The patterns o f inhibition observed support a mechanism where ATP binding precedes a minoglycoside binding followed by a rapid catalytic step, Product rele ase proceeds in an ordered fashion where kanamycin phosphate is releas ed quickly followed by a slow release of ADP. Aminoglycoside substrate s, such as kanamycin A, show substrate inhibition that is uncom petiti ve versus ATP. This indicates binding of the aminoglycosides to the sl owly dissociating (E . ADP) complex at high drug concentrations. These experiments are consistent with a Theorell-Chance kinetic mechanism f or 3'-aminoglycoside phosphotransferase-IIIa.