But‐3‐ene‐1,2‐diol: A Mechanism‐Based Active Site Inhibitor for Coenzyme B₁₂‐Dependent Glycerol Dehydratase

Abstract

Coenzyme B(12)-dependent glycerol dehydratase is a radical enzyme that catalyses the conversion of glycerol into 3-hydroxypropanal and propane-1,2-diol into propanal via enzyme-bound intermediate radicals. The substrate analogue but-3-ene-1,2-diol was studied in the expectation that it would lead to the 4,4-dihydroxylbut-2-en-1-yl radical, which is stabilised (allylic) and not reactive enough to retrieve a hydrogen atom from 5'-deoxyadenosine, thereby interrupting the catalytic cycle. Racemic and enantiomerically pure but-3-ene-1,2-diols and their [1,1-(2)H(2)], [2-(2)H] and [4,4-(2)H(2)] isotopomers were synthesised and characterised by NMR spectroscopy. (S)-[4-(14)C]but-3-ene-1,2-diol was also prepared. Kinetic measurements showed but-3-ene-1,2-diol to be a competitive inhibitor of glycerol dehydratase (K(i)=0.21 mM, k(i)=5.0x10(-2) s(-1)). With [4-(14)C]but-3-ene-1,2-diol it was demonstrated that species derived from the diol become tightly bound to the enzyme's active site, but not covalently bound, because the radioactivity could be removed upon denaturation of the enzyme. EPR measurements with propane-1,2-diol as substrate generated sharp signals after 10 s that disappeared after about 1 min. In contrast, EPR resonances appeared and disappeared more slowly when but-3-ene-1,2-diol was incubated with the enzyme. Among the deuterated isotopomers, only [1,1-(2)H(2)]but-3-ene-1,2-diol showed a significantly different EPR spectrum from that of the unlabelled diol; this indicated that coupling between the unpaired electron and a deuterium at C-1 was stronger than with deuterium at C-2 or C-4. The experiments suggest the formation of the 1,2-dihydroxybut-3-en-1-yl radical, which decomposes to unidentified product(s).

On the Reaction of Glycerol Dehydratase with But‐3‐ene‐1,2‐diol

“…[13] However, additional EPR studies with GDH and selectively deuterated but-3-ene-1,2-diols ( [ ]) suggested that the spin density during GDH inactivation is concentrated close to the C1 atom, rather than the C2 or C4 atoms. [10] This result was interpreted to indicate a build-up of the 1,2-dihydroxybut-3-en-1-yl radical (8). Relative to 9, however, 8 does not appear to possess any functionality that would render it particularly stable.…”

“…[10] It was found that DDH was also inactivated by the 3-unA C H T U N G T R E N N U N G sat-A C H T U N G T R E N N U N G urated diol in an EPR-active and time-dependent fashion. The identification of Ado À H by HPLC analysis implies that hydrogen abstraction from 7 did take place (step A in Equation (2)).…”

“…[10] The reaction demonstrated time-dependent inhibition of GDH to yield a complex that was spectroscopically (EPR) active. The incubation of GDH with (2S)- [4][5][6][7][8][9][10][11][12][13][14] C]-but-3-ene-1,2-diol suggested that one molecule of substrate binds per active site of GDH, and the purification procedure suggested that the species derived from but-3-ene-1,2-diol is not covalently bound to the active site of GDH.…”

“…[10] For example, the generation of 9 would be expected to render the ensuing hydrogen-transfer step (step C in Scheme 1) too demanding and would, thereby, terminate the catalytic cycle prematurely and irreversibly. Suicide inactivation mechanisms operating on similar principles have been described for both DDH [12a] and EAL.…”

“…[10] EPR spectral data for various isotopomers of 7 suggest that a signal arises from an interaction between cob(II)alamin and a species with spin density concentrated close to the C1 atom. On the basis of differences in the spectrum obtained with [ Initial H-atom abstraction from the C1 position of but-3-ene-1,2-diol: Scheme 3 displays some possible pathways for the GDH-catalyzed reaction when the initial H-atom abstraction occurs from the C1 position of but-3-ene-1,2-diol (7).…”