Moureau P, Engelborghs Y, Di Giambattista M, Cocito C
J Biol Chem. 1983 Dec 10;258(23):14233-8.
The kinetics of the interaction between the 50 S subunits (R) of bacterial ribosomes and the antibiotics virginiamycin S (VS), virginiamycin M (VM), and erythromycin have been studied by stopped flow fluorimetric analysis, based on the enhancement of VS fluorescence upon its binding to the 50 S ribosomal subunit. Virginiamycin components M and S exhibit a synergistic effect in vivo, which is characterized in vitro by a 5- to 10-fold increase of the affinity of ribosomes for VS, and by the loss of the ability of erythromycin to displace VS subsequent to the conformational change (from R to R*) produced by transient contact of ribosomes with VM. Our kinetic studies show that the VM-induced increase of the ribosomal affinity for VS (KVS = 25 X 10(6) M-1 instead of KVS = 5.5 X 10(6) M-1) is due to a decrease of the dissociation rate constant (k-VS = 0.008 s-1 instead of 0.04 s-1). The association rate constant remains practically the same (k+VS approximately k*+VS = 2.8 X 10(5) M-1 s-1), irrespective of the presence of VM. VS and erythromycin bind competitively to ribosomes. This effect has been exploited to determine the dissociation rate constant of VS directly by displacement experiments from VS . 50 S complexes, and the association rate constant of erythromycin (k+Ery = 3.2 X 10(5) M-1 S-1) on the basis of competition experiments for binding of free erythromycin and VS to ribosomes. By making use of the change in competition behavior of erythromycin and VS, after interaction of ribosomes with VM, the conformational change induced by VM has been explored. Within the experimentally available concentration region, the catalytic effect of VM has been shown to be coupled to its binding kinetics, and the association rate constant of VM has been determined (k+VM = 1.4 X 10(4) M-1 S-1). Evidence is presented for a low affinity binding of erythromycin (K*Ery approximately 3.3 X 10(4) M-1) to ribosomes altered by contact with VM. A model involving a sequence of 5 reactions has been proposed to explain the replacement of ribosome-bound erythromycin by VS upon contact of 50 S subunits with VM.
基于维及霉素S（VS）与50 S核糖体亚基结合时荧光增强，运用停流荧光分析法研究了细菌核糖体50 S亚基（R）与抗生素维及霉素S、维及霉素M（VM）及红霉素之间相互作用的动力学。维及霉素M和S组分在体内呈现协同效应，体外表现为核糖体对VS的亲和力增加5至10倍，以及核糖体与VM短暂接触产生构象变化（从R变为R*）后红霉素取代VS的能力丧失。我们的动力学研究表明，VM诱导的核糖体对VS亲和力增加（KVS = 25×10⁶ M⁻¹而非KVS = 5.5×10⁶ M⁻¹）是由于解离速率常数降低（k-VS = 0.008 s⁻¹而非0.04 s⁻¹）。无论VM是否存在，缔合速率常数基本保持不变（k+VS ≈ k*+VS = 2.8×10⁵ M⁻¹ s⁻¹）。VS和红霉素竞争性结合核糖体。利用这一效应，通过从VS·50 S复合物中进行置换实验直接测定VS的解离速率常数，并基于游离红霉素和VS与核糖体结合的竞争实验测定红霉素的缔合速率常数（k+Ery = 3.2×10⁵ M⁻¹ s⁻¹）。通过利用核糖体与VM相互作用后红霉素和VS竞争行为的变化，探索了VM诱导的构象变化。在实验可得的浓度范围内，已证明VM的催化作用与其结合动力学相关，并测定了VM的缔合速率常数（k+VM = 1.4×10⁴ M⁻¹ s⁻¹）。有证据表明，红霉素与因接触VM而改变的核糖体存在低亲和力结合（K*Ery ≈ 3.3×10⁴ M⁻¹）。已提出一个涉及5个反应序列的模型来解释50 S亚基与VM接触时VS取代核糖体结合的红霉素的过程。
