Abstract
The catalytic mechanism of Propionibacterium acnes polyunsaturated fatty acid isomerase (PAI) is explored by kinetic, spectroscopic, and thermodynamic studies. The PAI-catalyzed double bond isomerization takes place by selective removal of the pro-R hydrogen from C-11 followed by suprafacial transfer of this hydrogen to C-9 as shown by conversion of C-9-deuterated substrate isotopologs. Data on the midpoint potential, photoreduction, and cofactor replacement suggest PAI to operate via an ionic mechanism with the formation of FADH(2) and linoleic acid carbocation as intermediates. In line with this proposal, no radical intermediates were detected neither by stopped flow absorption nor by EPR spectroscopy. The substrate preference toward free fatty acids is determined by the interaction between Arg-88 and Phe-193, and the reaction rate is strongly affected by replacement of these amino acids, indicating that the efficiency of the hydrogen transfer relies on a fixed distance between the free carboxyl group and the N-5 atom of FAD. Combining data obtained for PAI from the structural studies and experiments described here suggests that at least two different prototypical active site geometries exist among polyunsaturated fatty acid double bond isomerases.
Highlights
Fatty acid (PUFA) double bond isomerases [4]
Hydrogen Transfer during the Isomerization—To establish the stereochemistry of hydrogen transfer during Propionibacterium acnes (PAI)-catalyzed isomerization, the reaction products deriving from LA were analyzed by GC-MS after conversion to 4,4-dimethyloxazoline derivatives
When [(11S)-2H]linoleic acid was used as a substrate, deuterium remained at position C-11, as indicated by ϩ1-atomic mass unit increase in the C-11 fragment, which is in line with the mechanism based on the PAI crystal structure [7]
Summary
Undeuterated molecules as determined by GC-MS analysis of the methyl ester derivative. Measurement 5 l of FA stock solution was mixed with buffer, Labeled Substrate Synthesis—[11,11-2H2]LA was prepared 0.5– 4 g of purified wt or mutant PAI protein added, and by organic synthesis using [1,1-2H2]1-bromo-2-octyne as the absorbance of the solution at 234 nm was recorded with an labeled synthon. Washing step was repeated twice; final protein concentration was about 1 mg/ml or 20 M With this preparation UV-visible spectra were recorded and activity tests performed. For the photoreduction of free PAI and PAI in complex with (10E,12Z)-CLA, 2 M 5-deaza-FAD and 1 M of the following redox dyes were added: methyl viologen (E ϭ Ϫ430 mV), 2-hydroxy-1,4-napthaquinone (E ϭ Ϫ145 mV), and phenazine methosulfate (E ϭ 80 mV). A total of 100 spectra were recorded successively in two different time regimes (50 spectra from 2 to 100 ms, 50 spectra from 100 ms to 100 s)
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