Slow-onset inhibition of ribosomal peptidyltransferase by lincomycin
| Autor: | Sofia Kallia-Raftopoulos, Charalambos Coutsogeorgopoulos, Dimitrios L. Kalpaxis |
|---|---|
| Rok vydání: | 1992 |
| Předmět: |
Poly U
Conformational change Stereochemistry Kinetics Biophysics RNA Transfer Amino Acyl Biochemistry Binding Competitive Non-competitive inhibition medicine Escherichia coli Molecular Biology Ternary complex Chemistry Ribosomal RNA Models Theoretical Lincomycin Crystallography Peptidyl Transferases Puromycin Isomerization Ribosomes Mathematics medicine.drug Logarithmic form |
| Zdroj: | Archives of biochemistry and biophysics. 298(2) |
| ISSN: | 0003-9861 |
| Popis: | 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 ⇌ K S CS → 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 k S obs = k 3 [ S ] (K s + [ S ]) . In the presence of lincomycin the logarithmic time plot is not a straight line, but becomes biphasic, giving an early slope ** K e = k 3 [ S ] {K s (1+[ I ] K 1 ) and a late slope ** k 1 = k 3 [ S ]{ K s (1+[ I ] k′ 1 +[ S ]} . Kinetic analysis of the early slopes at various concentrations of S and I shows competitive inhibition with Ki = 10.0 μM. The late slopes also give competitive inhibition with a distinct inhibition constant K'i = 2.0 μM. Excluding alternative models, the two phases of inhibition are compatible with a model in which reaction [1] is coupled with reaction [2], C + I ⇌ k 5 k 4 CI ⇌ k 7 k 6 C ∗ I where the isomerization step CI ⇌ CI∗ is slower than the first step C + I ⇌ CI, K i = k 5 k 4 and K′ i = K′ i [ k 7 (k 6 + k 7 ) ] . 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 eq I = k 6 + k 6 [I] (K i + [ I] ) . The plot of keqI versus [I] is not linear. This plot supports the two-step mechanism of reaction [2] in which k = 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. |
| Databáze: | OpenAIRE |
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