Evidence for substrate channeling in the early steps of cholesterogenesis.

3-Hydroxy-3-methylglutaryl-coenzyme A Synthase Reaction Intermediates: Detection of a Covalent Tetrahedral Adduct by Differential Isotope Shift 13C Nuclear Magnetic Resonance Spectroscopy

“…[l,3,5- 13 C] HMG-CoA. [1,3,5-13 C]HMG-CoA was enzymatically synthesized by condensing [1,[3][4][5][6][7][8][9][10][11][12][13] C]acetoacetyl-CoA with [1- 13 C]acetyl-CoA in the presence of catalytic amounts of purified recombinant avian HMG-CoA synthase (16). [1,[3][4][5][6][7][8][9][10][11][12][13] C]Acetoacetyl-CoA (13 µmol) and [1- 13 C]acetyl-CoA (12 µmol) were incubated with 1 mg of HMG-CoA synthase (1 unit/mg) at 30 °C in 100 mM Tris-HCl, pH 8.2, containing 0.10 mM EDTA.…”

“…This incorporates one 18 O into the HMG-CoA's free carboxylate, which should exhibit a C-O bond order of 1.5 due to resonance averaging of one single and one double bond. Proton-decoupled 13 C NMR spectra of [l,3,5-13 C]HMG-CoA synthesized in H 2 18 O vs H 2 16 O are shown in Figure 2A. Substitution of H 2 18 O for H 2 16 O resulted in an upfield shift of 0.026 ( 0.005 ppm for C5 (182.25 ppm) of [l,3,5-13 C]HMG-CoA, as measured after 5 h of signal accumulation (Table 1).…”

“…Proton-decoupled 13 C NMR spectra of [l,3,5-13 C]HMG-CoA synthesized in H 2 18 O vs H 2 16 O are shown in Figure 2A. Substitution of H 2 18 O for H 2 16 O resulted in an upfield shift of 0.026 ( 0.005 ppm for C5 (182.25 ppm) of [l,3,5-13 C]HMG-CoA, as measured after 5 h of signal accumulation (Table 1). The magnitude of the observed upfield shift is within the range expected for a C-O bond order of 1.5 (9).…”

“…However, at this time the hypothesis of the tetrahedral intermediate formation in the process of enzyme acetylation is based largely on solid bioorganic precedent (7) rather than direct experimental observations. The substitution of heavy stable isotopes (e.g., D for H, 18 O for 16 O) in metabolites produces small upfield shifts of the NMR signal of the carbon to which they are covalently linked (8,9). When deuterium is substituted for hydrogen, the observed effects are largest when the deuteron is directly bonded to the 13 C atom (R-effect) and are rapidly attenuated if the deuteron is separated by two (β-effect) or three (γeffect) bonds (8,10).…”

Kinetic fluorescence measurement of fluorescein di-.beta.-D-galactoside hydrolysis by .beta.-galactosidase: intermediate channeling in stepwise catalysis by a free single enzyme

“…Intermediate distribution and lifetime are of a great interest for understanding the kinetics of a multistep enzymatic reactions (Friedrich, 1984). In the immobilized or organized multienzyme systems, the concentrations of an intermediate in the immediate vicinity of the component enzymes were found to be greater than that in the bulk solution and the overall system reaction rates were always higher than those expected by the overall available concentrations of intermediate (Mosbach, 1976;Rugh, 1982; Gaertner, 1978;Matchett, 1974;Vitto et al, 1980; Akiyama & Hammes, 1981; Miziorko et al, 1990;Dunn et al, 1990;Sumegi et al, 1990). Recently, bifunctional complexes of ¡8-galactosidase-galactokinase (Billow et al, 1985;Billow, 1987) and /3-galactosidase-galactose dehydrogenase (Ljungcrantz et al, 1989) were prepared by means of an artificial gene fusion.…”