The kinetic mechanism of kanamycin acetyltransferase derived from the use of alternative antibiotics and coenzymes

• Published in: The Journal of biological chemistry Vol. 259; no. 20; pp. 12543 - 12546
• Main Authors: Radika, K, Northrop, D B
• Format: Journal Article
• Language: English
• Published: Bethesda, MD Elsevier Inc 25.10.1984; American Society for Biochemistry and Molecular Biology
• Subjects: Acetyltransferases - biosynthesis; Acetyltransferases - metabolism; Aminoglycosides - pharmacology; Anti-Bacterial Agents - pharmacology; Antibacterial agents; Antibiotics. Antiinfectious agents. Antiparasitic agents; Biological and medical sciences; Drug Resistance, Microbial; Enzyme Induction; Escherichia coli - enzymology; Kinetics; Medical sciences; Methylglycosides - analysis; Methylmannosides - analysis; Pharmacology. Drug treatments; Antibiotic; Structure activity relation; Kanamycine; Aminoglycoside
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)90781-7

Kinetic data for the 6‘-aminoglycoside-modifying enzyme, AAC(6‘)-4, also named kanamycin acetyltransferase, have been collected for six aminoglycoside antibiotics (amikacin, gentamicin C1a, kanamycin A, neomycin B, sisomicin, and tobramycin) and three coenzymes (acetyl-CoA, n-propionyl-CoA, and n-butyryl-CoA). The initial velocity pattern using acetyl-CoA favors a ping-pong kinetic mechanism (Kia = -0.34 +/- 0.34 microM), but the pattern using n-propionyl-CoA supports a sequential one (Kia = 2.7 +/- 0.8 microM). Kinetic analyses using alternative substrates confirm the sequential mechanism and, moreover, clearly identify a random order of addition of antibiotic and coenzyme because V/K values of antibiotics varied 40-fold as the identity of the coenzyme was changed and V/K values of coenzyme varied 13-fold as the identity of the antibiotic was changed. One or both sets of values would have remained unchanged if the mechanism were either ordered sequential or ping-pong. Combining these results with structure-activity data which argue for a rapid rate of release of substrates and products relative to the rate of enzymatic turnover (Radika, K., and Northrop, D. B. (1984) Biochemistry 25, in press) establishes the kinetic mechanism as rapid equilibrium random sequential.