Krekel F, Samland A K, Macheroux P, Amrhein N, Evans J N
School of Molecular Biosciences, Washington State University, Pullman, Washington 99164-4660, USA.
Biochemistry. 2000 Oct 17;39(41):12671-7. doi: 10.1021/bi001310x.
The enzyme UDP-N-acetylglucosamine (UDP-NAG) enolpyruvyltransferase (MurA) catalyzes the formation of enolpyruvyl-UDP-NAG, a precursor in peptidoglycan biosynthesis. The residue at position 115 in MurA has been proposed to act as a general acid in the enzymatic reaction. This is also the primary site of action of the antibiotic fosfomycin. In this paper, the pK(a) of Cys-115 has been determined to be 8.3, by titration of Enterobacter cloacae MurA with the alkylating agent iodoacetamide as a function of pH. Use of site-directed mutagenesis has established that only C115 is essential for catalysis, and the three other cysteine residues (C251, C354, and C381) are nonessential. Mass spectrometric analysis demonstrated that C115 is not alkylated at pH <7, but is alkylated significantly at pH >7. Measurement of the enzymatic inhibition by iodoacetamide as a function of pH showed maximum inhibition at pH >9, with a second-order rate constant of inhibition of 44 M(-)(1) s(-)(1) at pH 10. The presence of either one of the substrates did not influence the inactivation behavior, while the presence of both substrates resulted in a 5-fold reduction in the extent of alkylation. The covalent species that results from PEP bound to C115 of MurA exhibited 50-100-fold increased resistance against alkylation by iodoacetamide. These results imply that C115 is appreciably protonated at physiological pH and, therefore, is capable of acting as a proton donor in the enzyme-catalyzed reaction. However, it also implies that C115 is appreciably deprotonated at physiological pH also, whereupon the resultant thiolate nucleophile may play an important role in the formation of the covalent O-phosphothioketal species, whose role in catalysis is yet to be established.
酶UDP-N-乙酰葡糖胺（UDP-NAG）烯醇丙酮酸转移酶（MurA）催化烯醇丙酮酸-UDP-NAG的形成，烯醇丙酮酸-UDP-NAG是肽聚糖生物合成中的一种前体。MurA中第115位的残基被认为在酶促反应中起广义酸的作用。这也是抗生素磷霉素的主要作用位点。在本文中，通过用烷基化剂碘乙酰胺滴定阴沟肠杆菌MurA作为pH的函数，确定Cys-115的pK(a)为8.3。定点诱变的应用已确定只有C115对催化是必不可少的，而其他三个半胱氨酸残基（C251、C354和C381）则不是必需的。质谱分析表明，C115在pH<7时不被烷基化，但在pH>7时被显著烷基化。测量碘乙酰胺作为pH的函数对酶的抑制作用表明，在pH>9时抑制作用最大，在pH 10时抑制的二级速率常数为44 M⁻¹ s⁻¹。任何一种底物的存在都不会影响失活行为，而两种底物都存在则导致烷基化程度降低5倍。由与MurA的C115结合的PEP产生的共价物种对碘乙酰胺烷基化的抗性增加了50-100倍。这些结果表明，C115在生理pH下明显质子化，因此能够在酶催化反应中作为质子供体。然而，这也意味着C115在生理pH下也明显去质子化，于是由此产生的硫醇盐亲核试剂可能在共价O-磷硫酮物种的形成中起重要作用，其在催化中的作用尚待确定。
