PMID:7727397
| Citation |
Dhalla, AM, Yanchunas, J Jr, Ho, HT, Falk, PJ, Villafranca, JJ and Robertson, JG (1995) Steady-state kinetic mechanism of Escherichia coli UDP-N-acetylenolpyruvylglucosamine reductase. Biochemistry 34:5390-402 |
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| 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. |
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| Keywords |
Anaerobiosis; Carbohydrate Dehydrogenases/biosynthesis; Carbohydrate Dehydrogenases/isolation & purification; Carbohydrate Dehydrogenases/metabolism; Cloning, Molecular; Escherichia coli/enzymology; Escherichia coli/genetics; Flavin-Adenine Dinucleotide/metabolism; Genes, Bacterial; Glucosamine/analogs & derivatives; Glucosamine/metabolism; Kinetics; Mathematics; NADP/metabolism; Recombinant Proteins/isolation & purification; Recombinant Proteins/metabolism; Ribonucleotides/pharmacology; Uridine Diphosphate/analogs & derivatives; Uridine Diphosphate/metabolism; Uridine Diphosphate N-Acetylglucosamine/analogs & derivatives |
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