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., & 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.(ABSTRACT TRUNCATED AT 250 WORDS)