Slow-onset inhibition of ribosomal peptidyltransferase by lincomycin

In a system derived from Escherichia coli, we carried out a detailed kinetic analysis of the inhibition of the puromycin reaction by lincomycin. N-Acetylphenylalanyl-tRNA (Ac-Phe-tRNA; the donor) reacts with excess puromycin (S) according to reaction [1], C+S Ks <--> CS k3 --> C'+P, where C is the Ac-Phe-tRNA-poly(U)-ribosome ternary complex (complex C). The entire course of reaction [1] appears as a straight line when the reaction is analyzed as pseudo-first-order and the data are plotted in a logarithmic form (logarithmic time plot). The slope of this straight line gives the apparent ksobs = k3[S]/(Ks + [S]). In the presence of lincomycin the logarithmic time plot is not a straight line, but becomes biphasic, giving an early slope (ke = k3[S]/(Ks(1 + [I]/Ki) + [S])) and a late slope (k1 = k3[S]/(Ks(1 + [I]/K'i + [S])). Kinetic analysis of the early slopes at various concentrations of S and I shows competitive inhibition with Ki = 10.0 microM. The late slopes also give competitive inhibition with a distinct inhibition constant K'i = 2.0 microM. Excluding alternative models, the two phases of inhibition are compatible with a model in which reaction [1] is coupled with reaction [2], C+I k4 <--> k5 CI k6 <--> k7 C*I, where the isomerization step CI <--> CI* is slower than the first step C+I <--> CI, Ki = k5/k4 and K'i = Ki [k7/(k6 + k7)]. Corroborative evidence for this model comes from the examination of reaction [2] alone in the absence of S. This reaction is analyzed as pseudo-first-order going toward equilibrium with kIeq = k7 + (k6 [I]/(Ki + [I])). The plot of kIeq versus [I] is not linear. This plot supports the two-step mechanism of reaction [2] in which k6 = 5.2 min-1 and k7 = 1.3 min-1. This is the first example of slow-onset inhibition of ribosomal peptidyltransferase which follows a simple model leading to the determination of the isomerization constants k6 and k7. We suggest that lincomycin inhibits protein synthesis by binding initially to the ribosome in competition with aminoacyl-tRNA. Subsequently, as a result of a conformational change, an isomerization occurs (CI <--> C*I), after which lincomycin continues to interfere with the binding of aminoacyl-tRNA to the isomerized complex.