Determination of the pK(a) value of C115 in MurA (UDP-N-acetylglucosamine enolpyruvyltransferase) from Enterobacter cloacae

The enzyme UDP-N-acetylglucosamine (UDP-NAG) enolpyruvyltransferase (MurA) catalyzes the formation of enolpyruvyl-UDP-NAG, a precursor in peptidoglyca n biosynthesis. The residue at position 115 in MurA has been proposed to ac t as a general acid in the enzymatic reaction. This is also the primary sit e of action of the antibiotic fosfomycin. In this paper, the pK(a) of Cys-1 15 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-d irected mutagenesis has established that only C115 is essential for catalys is, and the three other cysteine residues (C251, C354, and C381) are noness ential. Mass spectrometric analysis demonstrated that C115 is not alkylated at pH <7, but is alkylated significantly at pH >7. Measurement of the enzy matic inhibition by iodoacetamide as a function of pH showed maximum inhibi tion 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 influenc e the inactivation behavior, while the presence of both substrates resulted in a 5-fold reduction in the extent of alkylation. The covalent species th at results from PEP bound to C115 of MurA exhibited 50-100-fold increased r esistance against alkylation by iodoacetamide. These results imply that C11 5 is appreciably protonated at physiological pH and, therefore, is capable of acting as a proton donor in the enzyme-catalyzed reaction. However, it a lso 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 ca talysis is yet to be established.