MECHANISM OF ACTION OF BUTYRYL-COENZYME A DEHYDROGENASE REACTIONS WITH ACETYLENIC, OLEFINIC AND FLUORINATED SUBSTRATE ANALOGS

Thioesters of acetylenic, olefinic and fluorinated acids were synthezised as mechanistic probes for the flavoenzyme butyryl-CoA dehydrogenase (EC 1.3.99.2) from Megasphaera elsdenii. These substrate analogs were examined with the enzyme to obtained evidence, that the enzymic dehydrogenation reaction is initiated by proton abstraction. In addition, it was hoped to identify a functional group at the active site.;Two other substrate analogs were found to inactivate butyryl-CoA dehydrogenase. 3-Chloro-3-butenoylpantetheine irreversibly inactivates the enzyme in a fashion similar to the acetylenic thioester and also modifies the (gamma)-carboxyl group of a glutamic acid. 2-Pentynoylpantetheine is also an irreversible inactivator of the enzyme. The inactivator does not form an adduct with the enzyme flavin and probably reacts with a nucleophilic group at the active site.;Butyryl-CoA dehydrogenase was found to catalyze an allylic isomerization as well as (beta)-elimination reactions. The enzyme isomerizes 3-butenoylpantetheine. With {2-('2)H(,2)}-3-butenoylpantetheine as the substrate 0.2 (+OR-) 0.05 deuterons are transferred to the (gamma)-carbon of the product. The enzyme catalyzes the elimination of HF from 3-fluoropropionyl-CoA and 3,3-difluorobutyrylpantetheine.;These results together show, that butyryl-CoA dehydrogenase has the capability to abstract protons. We suggest, that (alpha)-proton abstraction is a possible initial step in the dehydrogenation of acyl-CoA ester catalyzed by butyryl-CoA dehydrogenase.;The acetylenic thioester 3-pentynoylpantetheine irreversibly inactivates butyryl-CoA dehydrogenase. Upon inactivation, the enzyme flavin remains oxidized and can be reduced by dithionite. Enzyme maintained in the reduced state can be inactivated by 3-pentynoylpantetheine. {1-('14)C}-3-Pentynoylpantetheine becomes covalently attached to the protein (0.61 (+OR-) 0.1 mol ('14)C-inactivator/mol enzyme flavin). No modification of the flavin cofactor is seen. The covalent enzyme-inactivator adduct is labile towards base and neutral hydroxylamine. These treatments release 85 (+OR-) 5% of the incorporated ('14)C-label from the protein. Base catalyzed hydrolysis of the adduct releases 3-ketopentanoic acid (0.6 mon/mol incorporated inactivator). Treatment with hydroxylamine leads to formation of a hydroxamic acid on the protein (0.64 (+OR-) 0.09 mol/mol incorporated inactivator). The covalent adduct can be reduced with sodium borohydride with release of 1,3-pentanediol. Hydrolysis of the protein with 6 N HCl after sodium borohydride reduction yields 2-NH(,2)-5-OH-valeric acid and proline. We conclude that the inactivator has reacted with the (gamma)-carboxyl group of a glutamate residue at the active site. The inactivation proceeds through enzyme catalyzed rearrangment of the acetylene to the allene.