title: Steady-state kinetic mechanism of Escherichia coli UDP-N-acetylenolpyruvylglucosamine reductase.
abstract: The Escherichia coli MurB gene encoding UDP-N-acetylenolpyruvylglucosamine reductase was expressed to a level of approximately 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 KiS were 910 microM and 73 microM. 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 Km,UNAGEP = 24 +/- 3 microM, Ki,UNAGEP = 73 +/- 19 microM, Km,NADPH = 17 +/- 3 microM, Ki,NADPH = 910 +/- 670 microM, and kcat = 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 microM. 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.
authors.affiliation: Enzymology Laboratory, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543-4000, USA.
authors.forename: A M
authors.initials: AM
authors.lastname: Dhalla
authors.forename: J
authors.initials: J
authors.lastname: Yanchunas
authors.forename: H T
authors.initials: HT
authors.lastname: Ho
authors.forename: P J
authors.initials: PJ
authors.lastname: Falk
authors.forename: J J
authors.initials: JJ
authors.lastname: Villafranca
authors.forename: J G
authors.initials: JG
authors.lastname: Robertson
mesh_terms: D000693:Anaerobiosis; D002237:Carbohydrate Dehydrogenases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D005798:Genes, Bacterial; D005944:Glucosamine / Q000031:analogs & derivatives / Q000378:metabolism; D007700:Kinetics; D008433:Mathematics; D009249:NADP / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism*; D012265:Ribonucleotides / Q000494:pharmacology; D014530:Uridine Diphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D014537:Uridine Diphosphate N-Acetylglucosamine / Q000031:analogs & derivatives*
chemical_list: D011994:Recombinant Proteins; D012265:Ribonucleotides; D005182:Flavin-Adenine Dinucleotide; D014537:Uridine Diphosphate N-Acetylglucosamine; D009249:NADP; D014530:Uridine Diphosphate; C038277:UDP-N-acetylglucosamine-enolpyruvate; D002237:Carbohydrate Dehydrogenases; C023215:UDP-N-acetylmuramate dehydrogenase; D005944:Glucosamine