Kim D H, Lees W J, Kempsell K E, Lane W S, Duncan K, Walsh C T
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Biochemistry. 1996 Apr 16;35(15):4923-8. doi: 10.1021/bi952937w.
The antibiotic fosfomycin inhibits bacterial cell wall biosynthesis by inactivation of UDP-GlcNAc enolpyruvyl tranferase (MurA). Prior work has established that Cys115 of Escherichia coli and Enterobacter cloacae MurA is the active site nucleophile alkylated by fosfomycin and implicated this residue in the formation of a covalent phospholactyl-enzyme adduct derived from substrate, phosphoenolpyruvate (PEP). On the basis of sequencing information from putative MurA homolog from Mycobacterium tuberculosis, we generated a C115D mutant of E. coli MurA that was highly active but fully resistant to time-dependent inhibition by fosfomycin. Fosfomycin still bound to the active site of C115D MurA, as established by the observed reversible competitive inhibition by fosfomycin. Fosfomycin still bound to the active site of C115D MurA, as established by the observed reversible competitive inhibition vs PEP. In contrast to the broad pH-independent behavior of wild-type (WT) MurA, C115D mutant activity titrated across the pH range examined (pH 5.5-9) with an apparent pKa approximately 6, with kcatC115D ranging from approximately 10kcatWT at pH 5.5 to <0.1kcatWT at pH9.0. Km(PEP)115D was relatively constant in the pH range examined and increased approximately 100-fold relative to Km(PEP)WT. A fosfomycin-resistant C115E mutant with -1% activity of the C115D mutant was found to follow a pH dependence similar to that observed for C115D MurA. The contrasting pH dependences of WT and C115D MurA was also observed in the reaction with the pseudosubstrate, (Z)-3-fluorophosphoenolpyruvate, strongly suggesting a role for Cys/Asp115 as the general acid in the protonation of C-3 of PEP during MurA-catalyzed enol ether transfer. The difference in nucleophilicity between the carboxylate side chains of Asp115 and Glu115 and the thiolate group of Cys115 suggests that covalent enzyme adduct formation is not required for catalytic turnover and, furthermore, provides a chemical rationale for the resistance of the C115D and C115E mutants to fosfomycin inactivation.
抗生素磷霉素通过使UDP-N-乙酰葡糖胺烯醇丙酮酸转移酶（MurA）失活来抑制细菌细胞壁的生物合成。先前的研究已证实，大肠杆菌和阴沟肠杆菌MurA的Cys115是被磷霉素烷基化的活性位点亲核试剂，并认为该残基参与了由底物磷酸烯醇丙酮酸（PEP）形成的共价磷酰基酶加合物的过程。基于来自结核分枝杆菌推定的MurA同源物的测序信息，我们构建了大肠杆菌MurA的C115D突变体，该突变体具有高活性，但对磷霉素的时间依赖性抑制具有完全抗性。正如磷霉素所观察到的可逆竞争性抑制所证实的那样，磷霉素仍然结合到C115D MurA的活性位点。与野生型（WT）MurA广泛的pH无关行为相反，C115D突变体的活性在所研究的pH范围（pH 5.5 - 9）内呈滴定变化，表观pKa约为6，kcatC115D范围从pH 5.5时约为10kcatWT到pH 9.0时<0.1kcatWT。Km(PEP)115D在所研究的pH范围内相对恒定，相对于Km(PEP)WT增加了约100倍。发现活性为C115D突变体-1%的磷霉素抗性C115E突变体遵循与C115D MurA观察到的类似的pH依赖性。在与假底物（Z）-3-氟磷酸烯醇丙酮酸的反应中也观察到了WT和C115D MurA相反的pH依赖性，这强烈表明Cys/Asp115在MurA催化的烯醇醚转移过程中作为使PEP的C-3质子化的一般酸发挥作用。Asp115和Glu115的羧酸盐侧链与Cys115的硫醇盐基团之间亲核性的差异表明，催化周转不需要共价酶加合物的形成，此外，还为C115D和C115E突变体对磷霉素失活的抗性提供了化学原理。
