STEADY-STATE KINETIC MECHANISM OF ESCHERICHIA-COLI UDP-N-ACETYLENOLPYRUVYLGLUCOSAMINE REDUCTASE

The Escherichia coli MurB gene encoding UDP-N-acetylenolpyruvylglucosamine reductase was expressed to a level of approximate to 100 mg/L as a fusion construct with maltose binding protein. Rapid affinity purification, proteolysis, and anion exchange chromatography yielded homogeneous enzyme containing 1 mol/mol bound FAD. Enzyme was maximally activated by K+, NH4+, and Rb+ at cation concentrations between 10 and 50 mM. Steady-state enzyme kinetics at pH 8.0 and 37 degrees C revealed weak and strong substrate inhibition by NADPH and UDP-N-acetylenolpyruvylglucosamine, respectively, where the K(i)s were 910 mu M and 73 mu M. Substrate inhibition was pH dependent for both substrates. Initial velocity measurements as a function of both substrates produced patterns consistent with a ping pong bi bi double competitive substrate inhibition mechanism. Data at pH 8.0 yielded kinetic constants corresponding to K-m,K-UNAGEP = 24 +/- 3 mu M, K-i,K-UNAGEP = 73 +/- 19 mu M, K-m,K-NADPH = 17 +/- 3 mu M, K-i,K-NADPH = 910 +/- 670 mu M, and k(cat) = 62 +/- 3 s(-1). A slow anaerobic exchange reaction with thio-NADP(+) provided evidence for release of NADP(+) in the absence of UNAGEP. Alternate reduced nicotinamide dinucleotides, including NHXDPH, 3'-NADPH, and alpha-NADPH, were substrates, whereas NADH was not. Several nucleotides, including ADP and UDP, were weak inhibitors of the enzyme with inhibition constants between 5 and 97 mM. Various analogs of NADP(+), including 3'-NADP(+), thio-NADP(+), APADP(+), NEthDP(+), and NHXDP(+), were inhibitors of the enzyme with respect to NADPH and yielded inhibition constants in the range of 110-1100 mu M. Analogs without the 2'- or 3'-phosphate of NADPH or NADP(+) were not substrates or inhibitors. Double inhibition experiments with varied APADP(+) and UNAG produced inhibition patterns consistent with mutually exclusive inhibitor binding. The data suggest that NADPH and UNAGEP share a subsite that prevents both molecules from binding at once.