Alternative substrate and inhibition kinetics of aminoglycoside nucleotidyltransferase 2''-I in support of a Theorell-Chance kinetic mechanism

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Abstract
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. and 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. and Northrop, D.B. (1984) Arch. Biochem Biophys. 230, 250-263]. This doubly broasd substrate specificity makes it an excellent candidate for application of the alternative substrate diagnostic [Radika, K., and 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 fasion 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. In a new and complex form of multiple inhibition, the substrate inhibition constant of tobramycin increases with increasing concentrations of dead-end inhibitor, signifying competition for EQ; but surprisingly, the dead-end inhibitor activates the substrate-inhibited enzyme at high concentrations, consistent with a more rapid escape of Q from EQI than from EQB. Product inhibition by AMP-tobramycin is noncompetitive versus Mg-ATP and uncompetitive versus tobramycin; in the latter, the substrate inhibition constant increases with increasing concentrations of product, signifying the formation of an EQQ complex. Moreover the lack of enhancement of substrate inhibition at moderate concentrations of added product confirms that the enzyme exists primarily as the EQ complex during normal turnover.