Bernat B A, Armstrong R N
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, USA.
Biochemistry. 2001 Oct 23;40(42):12712-8. doi: 10.1021/bi0114832.
The fosfomycin resistance protein, FosA, catalyzes the Mn(2+)-dependent addition of glutathione to the antibiotic fosfomycin, (1R,2S)-epoxypropylphosphonic acid, rendering the antibiotic inactive. The enzyme is a homodimer of 16 kDa subunits, each of which contains a single mononuclear metal site. Stopped-flow absorbance/fluorescence spectrometry provides evidence suggesting a complex kinetic mechanism for the acquisition of Mn(2+) by apoFosA. The binding of Mn(H(2)O)(6)(2+) to apoFosA alters the UV absorption and intrinsic fluorescence characteristics of the protein sufficiently to provide sensitive spectroscopic probes of metal binding. The acquisition of metal is shown to be a multistep process involving rapid preequilibrium formation of an initial complex with release of approximately two protons (k(obsd) > or = 800 s(-1)). The initial complex either rapidly dissociates or forms an intermediate coordination complex (k > 300 s(-1)) with rapid isomerization (k > or = 20 s(-1)) to a set of tight protein-metal complexes. The observed bimolecular rate constant for formation of the intermediate coordination complex is 3 x 10(5) M(-1) s(-1). The release of Mn(2+) from the protein is slow (k approximately 10(-2) s(-1)). The kinetic results suggest a more complex chelate effect than is typically observed for metal binding to simple multidentate ligands. Although the addition of the substrate, fosfomycin, has no appreciable effect on the association kinetics of enzyme and metal, it significantly decreases the dissociation rate, suggesting that the substrate interacts directly with the metal center.
磷霉素抗性蛋白FosA催化谷胱甘肽以锰离子依赖的方式加成到抗生素磷霉素（(1R,2S)-环氧丙基膦酸）上，使抗生素失活。该酶是由16 kDa亚基组成的同型二聚体，每个亚基包含一个单核金属位点。停流吸收/荧光光谱法提供的证据表明，脱辅基FosA获取锰离子的动力学机制较为复杂。Mn(H₂O)₆²⁺与脱辅基FosA的结合足以改变蛋白质的紫外吸收和固有荧光特性，从而提供金属结合的灵敏光谱探针。金属的获取过程显示为一个多步骤过程，包括与大约两个质子的释放一起快速形成初始复合物的预平衡（观测速率常数k(obsd)≥800 s⁻¹）。初始复合物要么迅速解离，要么形成一个中间配位复合物（速率常数k>300 s⁻¹），并快速异构化（速率常数k≥20 s⁻¹）为一组紧密的蛋白质-金属复合物。观测到的形成中间配位复合物的双分子速率常数为3×10⁵ M⁻¹ s⁻¹。锰离子从蛋白质中的释放很慢（速率常数k约为10⁻² s⁻¹）。动力学结果表明，与通常观察到的金属与简单多齿配体的结合相比，存在更复杂的螯合效应。尽管底物磷霉素的添加对酶与金属的缔合动力学没有明显影响，但它显著降低了解离速率，这表明底物直接与金属中心相互作用。
