Determination of the rate-limiting segment of aminoglycoside nucleotidyltransferase 2''-I by pH- and viscosity-dependent kinetics

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Abstract
Aminoglycoside nucleotidyltransferase 2''''-I follows a Theorell-Chance kinetic mechanism in which turnover is controlled by the rate-limiting release of the final product (Q), a nucleotidylated aminoglycoside [Gates, C. A., and Northrop, D. B. (1988) Biochemistry (second of three papers in this issue)]. The effects of viscosity on the kinetic constants of netilmicin, gentamicin C1, and sisomicin aminoglycoside substrates are as follows: no change in the substrate inhibition constants of all three antibiotics, a small but significant and highly unusual increase in Vmax/Km for netilmicin but large, normal decreases for gentamicin C1 and sisomicin, and marked decreases in the maximal velocities for all three. The lack of effect on substrate inhibition provides essential control experiments, signifying that glycerol does not interfere with binding of aminoglycosides to EQ and that the steady-state distribution of EQ does not increase as the release of Q is slowed by a viscosogen. The decrease in the Vmax/Km of better substrates indicates dominance by a diffusion-controlled component in the catalytic segment, attributed to the release of pyrophosphate. The presence of an increase in the Vmax/Km of the poor substrate, however, is inexplicable in terms of either single or multiple diffusion-controlled steps. Instead, it is here attributed to an equilibrium between conformers of the enzyme-nucleotide complex in which glycerol favors the conformation necessary for binding of aminoglycosides. The decrease in Vmax is consistent with the diffusion-controlled release of the final product determining enzymatic turnover. The substrate inhibition constant of sisomicin as a function of pH is a bell-shaped curve with asymptotes of slopes +2 and -2, representing two acidic and two alkaline ionizations, with pK values of 6.68 .+-. 0.08 and 8.22 .+-. 0.06, respectively. The pH profile of Vmax/Km is strikingly similar, with pK values of 6.76 .+-. 0.07 and 8.45 .+-. 0.05. This similarity signifies a common origin and indicates that the aminoglycoside substrate (B) binds to EA in identical fashion as to EQ and that catalysis is pH-independent. Binding identity requires that the aminoglycoside portion of Q does not occupy its subsite on the enzyme; rather, the aminoglycoside portion is released during catalysis, driven by the exothermic loss of a phosphate anhydride bond and stereochemical inversion of the .gamma.-phosphate [Van Pelt, J. E., Iyengar, R., and Frey, P. A. (1986) J. Biol. Chem. 261, 15995-15999]. The maximal velocity as a function of pH is described by a shallow wave with a pK of 8.4 .+-. 0.1. The pH-independent region of the Ki profile coincident with the pH-dependent region of the Vmax profile shows that the steady-state concentration of EQ is insensitive to changes in pH that affect turnover; thus the kinetic mechanism of the enzyme does not change with pH. The drastic dissimilarity between the pH profiles of Vmax/Km and Vmax confirms the Theorell-Chance kinetic mechanism because these two kinetic constants cannot hold any reactive steps in common.

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