SPECTROSCOPIC PROPERTIES OF ESCHERICHIA-COLI UDP-N-ACETYLENOLPYRUVYLGLUCOSAMINE REDUCTASE

Purified uridine diphosphate N-acetylenolpyruvylglucosamine reductase (E.C. 1.1.1.158) was analyzed by circular dichroism (CD) and UV-visibl e spectroscopy to establish the spectral properties of its tightly bou nd flavin adenine dinucleotide (FAD) cofactor. The polypeptide backbon e displayed a single circular dichroic minimum at 208 nm and a single maximum at 193 nm. The CD spectrum of bound flavin exhibited a single major negative Cotton peak at 364 nm and two minor negative Cotton pea ks at 464 and 495 nm, The protein was reversibly unfolded in 9.8 M ure a and refolded in buffer in the presence of excess FAD, The refolded e nzyme incorporated FAD and catalyzed full activity. The bound FAD disp layed an absorption maximum at 464 nm with an extinction coefficient o f epsilon(464) = 11700 M(-1) cm(-1). Anaerobic reduction with dithioni te was complete at 1 equiv. Anaerobic reduction with nicotinamide aden ine dinucleotide phosphate, reduced form (NADPH), also was essentially complete at 1 equiv and produced a long-wavelength absorbance band ch aracteristic of an FAD-pyridine nucleotide charge transfer complex. Ph otochemical bleaching in the presence of ethylenediaminetetraacetic ac id (EDTA) followed exponential kinetics, None of the anaerobic reducti ve titrations produced a spectral intermediate characteristic of a fla vin semiquinone, and all reduced enzyme species could be fully reoxidi zed by oxygen, with full recovery of catalytic activity. Photochemical ly reduced enzyme was reoxidized by titration with either NADP(+) or u ridine diphospho N-acetylglucosamine enolpyruvate (UNAGEP), Reoxidatio n by NADP(+) reached a chemical equilibrium, whereas reoxidation by UN AGEP was stoichiometric. Binding of NADP(+) or UNAGEP to the oxidized form of the enzyme produced a dead-end complex that could be titrated by following a 10-nm red shift in the absorption spectrum of the bound FAD. The K-d Of NADP(+) for oxidized enzyme was 0.7 +/- 0.3 mu M and the K-d Of UNAGEP was 2.7 +/- 0.3 mu M Solvent deuterium isotope effec ts on binding were observed for both NADP(+) and UNAGEP, depending on the pH. At pH 8.5, the K-H(d)/K-D(d) was 2.2 for NADP(+) and 3.9 for U NAGEP. No spectral changes were observed in the presence of a 40-fold excess of uridine diphospho N-acetylmuramic acid (UNAM) either aerobic ally or anaerobically. These studies have identified spectral signals for five steps in the kinetic mechanism, have indicated that product f ormation is essentially irreversible, and have indicated that hydrogen bonding or protonation contributes significantly to ground-state comp lex formation with the physiological substrate.