Alternative substrate and inhibition kinetics of aminoglycoside nucleotidyltransferase 2''-I in support of a Theorell-Chance kinetic mechanism.
Aminoglycoside nucleotidyltransferase 2''-I conveys multiple antibiotic resistance to Gram-negative bacteria because the enzyme adenylylates a broad range of aminoglycoside antibiotics as substrates [Gates, C. A., & Northrop, D. B. (1988) Biochemistry (preceding paper in this issue)]. The enzyme also catalyzes the transfer of a variety of nucleotides [Van Pelt, J. E., & Northrop, D. B. (1984) Arch. Biochem. Biophys. 230, 250-263]. This doubly broad substrate specificity makes it an excellent candidate for application of the alternative substrate diagnostic [Radika, K., & Northrop, D. B. (1984) Anal. Biochem. 141, 413-417] as a means to determine its kinetic mechanism. The kinetic patterns presented here are composed of one set of intersecting lines and one coincident line and are consistent with a Theorell-Chance kinetic mechanism in which nucleotide binding precedes aminoglycosides, pyrophosphate is released prior to the nucleotidylated aminoglycoside (Q), and turnover is controlled by the rate-limiting release of the final product. Substrate inhibition by tobramycin (B) is partial and uncompetitive versus Mg-ATP, indicating that B binds to the EQ complex, but not in the usual dead-end fashion common to an ordered sequential release of products; instead, Q may escape from the abortive EQB complex at a finite rate. Dead-end inhibition by neomycin C (I) is also partial and uncompetitive versus Mg-ATP but is slope-linear, intercept-hyperbolic, partial noncompetitive versus gentamicin A; both kinetic patterns signify the formation of a partial abortive EQI complex.(ABSTRACT TRUNCATED AT 250 WORDS)
Gates
C A
CA
Division of Pharmaceutical Biochemistry, School of Pharmacy, University of Wisconsin, Madison 53706.
Northrop
D B
DB
D000617:Aminoglycosides / Q000494:pharmacology; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001667:Binding, Competitive; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D008433:Mathematics; D008962:Models, Theoretical; D009713:Nucleotidyltransferases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013379:Substrate Specificity
D000617:Aminoglycosides; D000900:Anti-Bacterial Agents; D009713:Nucleotidyltransferases; C020565:gentamicin 2''-nucleotidyltransferase