Manganese (II) And spin-labeled uridine 5'-diphosphate binding to bovine galactosyltransferase.
The kinetically observed Mn(II) activation as well as inhibition has been clarified for bovine galactosyltransferase. An electron spin resonance (ESR) titration of MnCl2 with galactosyltransferase alone at pH 8.0 clearly shows the existence of at least two metal ion binding sites with microscopic dissociation constants of 0.84 +/- 0.1 and 9.0 +/- 1.0 mM, respectively. The second site corresponds with either published kinetic constant for Mn(II) of 8.5 mM (inhibition) or 3.40 mM (activation). The contribution of the binary complex Mn(II)-UDPGal is of lesser significance, as concluded by its ESR measured Kdiss of 14.5 +/- 1.1 mM at pH 8.0. A spin-labeled inhibitor analog of UDPgalactose, UDP-4-O-(2,2,6,6-tetramethyl-4-piperidinyl-1-oxy), or UDP-R, was synthesized as a competitive inhibitor for UDPGal. It was shown from inhibition kinetics to be almost as potent an inhibitor as UDPGlu. The Ki values at pH 8.0 in the N-acetyllactosamine and lactose reactions were 0.38 +/- 0.04 and 0.63 +/- 0.06 mM, respectively, as compared with 0.10 +/- 0.01 and 0.094 +/- 0.009 mM for UDPGlu. An ESR titration of UDP-R with galactosyltransferase at pH 8.0 yielded direct physical dissociation constants of 0.40 +/- 0.07 and 0.53 +/- 0.08 mM in the absence and presence of alpha-lactalbumin, respectively. No other substrates (glucose of N-acetylglucosamine) nor Mn(II) were present.
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D004578:Electron Spin Resonance Spectroscopy; D004789:Enzyme Activation / Q000187:drug effects; D005260:Female; D007700:Kinetics; D007788:Lactose Synthase / Q000378:metabolism; D008345:Manganese / Q000494:pharmacology; D008433:Mathematics; D008892:Milk / Q000201:enzymology; D011485:Protein Binding; D011487:Protein Conformation; D013113:Spin Labels; D014500:Uracil Nucleotides
D013113:Spin Labels; D014500:Uracil Nucleotides; D008345:Manganese; D007788:Lactose Synthase