UDP-galactose 4-epimerase: NAD+ content and a charge-transfer band associated with the substrate-induced conformational transition.
UDP-galactose 4-epimerase from Escherichia coli contains tightly bound NAD+, which participates in catalyzing the interconversion of UDP-galactose and UDP-glucose through its redox properties. The purified enzyme is a dimer of identical subunits that consists of a mixture of catalytically active subunits designated E.NAD+ and inactive, abortive complexes designated E.NADH.uridine nucleotide, in which the uridine nucleotide may be UDP-glucose, UDP-galactose, or UDP [Vanhooke, J. L., & Frey, P. A. (1994) J. Biol. Chem. 269, 31496-31404]. The abortive complexes are transformed into active E.NAD+ by denaturation of the purified enzyme at 4 degrees C in 6 M guanidine hydrochloride buffered at pH 7.0 in the presence of 0.126 mM NAD+ for 3 h, followed by dilution of guanidine hydrochloride to 0.18 M and of NAD+ to 0.076 mM for 2 h. The renatured enzyme is fully active and contains negligible amounts of NADH and uridine nucleotides. The extinction coefficent of the epimerase at 280 nm is 1.81 +/- 0.15 mL mg-1 cm-1 (epsilon 280 = 137 +/- 11 mM-1 cm-1), as determined by quantitative amino acid analysis and spectrophotometric measurements. This value allows the value of the extinction coefficient for the reduced enzyme (E.NADH)to be calculated as epsilon 344 = 5.7 mM-1 cm-1. On the basis of the new value of epsilon 280, analytical measurements of the nAD+ content of epimerase show that there are two molecules of NAD+ per dimer, which confirms conclusions from X-ray crystallography and revises the earlier bioanalytical determinations. The ultraviolet/visible absorption spectrum of E.NAD+ from denaturation-renaturation experiments reveals the presence of a broad absorption band extending from 300 nm to beyond 360 nm that cannot be attributed to NADH and appears to be a charge-transfer band. This band is partially bleached by UMP and almost totally abolished by UDP, indicating that the interactions leading to the charge-transfer band are altered by the uridine nucleotide-induced conformational change in this enzyme. This conformational change is associated with control of the chemical reactivity of NAD+ in the reaction mechanism.
Liu
Y
Y
Institute for Enzyme Research, Graduate School, Madison, Wisconsin 53705, USA.
Vanhooke
J L
JL
Frey
P A
PA
D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D009243:NAD / Q000378:metabolism / Q000494:pharmacology; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D014534:UDPglucose 4-Epimerase / Q000737:chemistry / Q000378:metabolism*; D014508:Urea / Q000494:pharmacology; D014530:Uridine Diphosphate / Q000378:metabolism
D011994:Recombinant Proteins; D009243:NAD; D014530:Uridine Diphosphate; D014508:Urea; D014534:UDPglucose 4-Epimerase