Slow sequential conformational changes in Escherichia coli ribosomes induced by lincomycin: kinetic evidence.

In a cell-free system derived from Escherichia coli, lincomycin produces biphasic logarithmic time plots for inhibition of peptide-bond formation when puromycin is used as an acceptor substrate and AcPhe-tRNA as a donor substrate. In a previous study, initial slope analysis of the logarithmic time plots revealed that the encounter complex CI between the initiator ribosomal complex (C) and lincomycin (I) undergoes a slow isomerization to C*I. During this change, the bound AcPhe-tRNA and lincomycin are rearranged to also accommodate puromycin, and this may account for the mixed noncompetitive inhibition (K(i)* = 70 microM) established at higher concentrations of the drug. The above-mentioned effect was further investigated by analyzing the late phase of the logarithmic time plots. It was found that C*I complex reacts with a second molecule of I, giving C*I(2) complex. However, the logarithmic time plots remain biphasic even at high concentrations of lincomycin, making possible the identification of another inhibition constant K(i)*', which is equal to 18 microM. The simplest explanation of this finding is to assume the existence of a second isomerization step C*I(2) <--> C*I(2'), slowly equilibrated. The determination of K(i)*' enables us to calculate the isomerization constant (K(isom) = 2.9) with the formula K(i)*' = K(i)*/(1 + K(isom)). Our results suggest that whenever a fast and reversible interaction of lincomycin with the elongating ribosomal complex C occurs, the latter undergoes a slow isomerization, which may be the result of conformational changes induced by the drug.

Sparsomycin and its analogues: a new approach for evaluating their potency as inhibitors of peptide bond formation.

The ability of several sparsomycin analogues to inhibit peptide bond formation was studied in vitro. Peptide bonds are formed between puromycin (S) and the acetylPhe-tRNA of acetylPhe-tRNA/70 S ribosome/poly(U) complex (complex C), according to the puromycin reaction: [formula: see text] It was shown that the sparsomycin analogues, like sparsomycin itself, inhibit peptide bond formation in a time-dependent manner; they react with complex C according to the equation [formula: see text] where C*I is a conformationally altered species in which I is bound more tightly than in CI. The determination of the rate constant k(7) for the regeneration of complex C from the C*I complex allows evaluation of these analogues as inhibitors of peptide bond formation. According to their k7 values, these analogues are classified in order of descending potency as follows: n-pentyl-sparsomycin (4) > n-butyl-sparsomycin (3) approximately n-butyl-deshydroxy-sparsomycin (6) > benzyl-sparsomycin (2) > deshydroxy-sparsomycin (5) approximately sparsomycin (1) > n-propyl-desthio-deshydroxy-sparsomycin (7). The analogues with an aromatic or a larger hydrophobic side chain are stronger inhibitors of the puromycin reaction than are those with a smaller side chain or those lacking the bivalent sulfur atoms; replacement of the hydroxymethyl group with a methyl group does not affect the position of the compound in this ranking; compare the positions of compounds 1 and 3 with those of 5 and 6. In the case of compound 7, C*I adsorbed on cellulose nitrate disks was not sufficiently stable to allow examination by the method applied to the other analogues, probably due to a relatively large value of k7. This analogue showed also time-dependent inhibition, but after the isomerization of CI to C*I, the kinetics of inhibition become complex, and C*I interacted further with puromycin, either as C*I or after its dissociation to C*.

New aspects of the kinetics of inhibition by lincomycin of peptide bond formation.

We have investigated the inhibition of peptide bond formation by the antibiotic lincomycin, at 150 mM NH4Cl. We have used an in vitro system in which a ribosomal ternary complex, the acetyl[3H] phenylalanine-tRNA-70 S ribosome-poly(U) complex (complex C), reacts with puromycin, forming peptide bonds. Complex C can be considered an analog of the elongating ribosomal complex and puromycin an analog of aminoacyl-tRNA. In a previous study we reported on the kinetics of inhibition by lincomycin at 100 mM NH4Cl. In the present investigation, we find that an increase of the ammonium ion concentration to 150 mM causes profound changes in the kinetic behavior of the system, which can be summarized as follows. First, the association rate for complex C and lincomycin is increased. At a lincomycin concentration of 10 microM the apparent equilibration rate constant is 4.3 min-1 at 100 mM NH4Cl, whereas it becomes 6.7 min-1 at 150 mM. Second, at 150 mM NH4Cl, with increasing concentrations of lincomycin, there is a transition from competitive to mixed-noncompetitive inhibition. The prevailing notion is that lincomycin acts at the ribosomal A-site, a mechanism that agrees only with competitive kinetics (mutually exclusive binding between puromycin and lincomycin). At the molecular level, the change in the kinetics of inhibition that we observe may mean that the mutually exclusive binding between aminoacyl-tRNA and lincomycin is converted to simultaneous binding, as a result of conformational changes occurring in the elongating ribosomal complex.

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.