Model studies of coenzyme B12 dependent reactions

Abstract

Coenzyme-B12 dependent glutamate mutase is a radical enzyme, which catalyses the isomerisation of (S)-glutamate to (2S,3S)-3-methylaspartate via enzyme-bound radical intermediates. This is the key step in the fermentation of glutamate to ammonia, carbon dioxide, acetate, butyrate and hydrogen by Clostridium tetanomorphum. It has been proposed that the mechanism of glutamate mutase involves removal of Hsi from glutamate to give a 4-glutamyl radical that undergoes fragmentation to acrylate and a glycine radical. Recombination of these species leads to the 3-methylene-aspartate radical and hence (2S,3S)-3-methylaspartate. Glutamate mutase has an active site containing three arginine residues that bind to the carboxylate groups of substrate, intermediates and product, the so-called ‘arginine claw’. The model compound containing two guanidinium groups, 2,3-bisguanidinomethyl- biphenyl dihydrochloride, has been synthesised in order to probe the function of this peculiar enzyme active site. NMR titration results showed that the receptor binds weakly to tetrabutylammonium glutarate in CD3OD. To identify possible intermediates for the glutamate mutase catalysed reaction, a precursor for the product-related radical 3-methyleneaspartate has been designed and its synthesis has been explored. Diol dehydratase from e.g. Klebsiella oxytoca is a radical enzyme that converts simple 1,2-diols into corresponding aldehydes and water. The enzyme requires adenosylcobalamin (coenzyme B12) as cofactor and a metal cation (e.g. K+). The mechanism of action of the dehydratase has previously been investigated by protein crystallography and ab initio molecular orbital calculations, aided by stereochemical and model studies. The 5'-deoxyadenosyl radical from homolysis of the coenzyme’s Co-C bond abstracts a specific H atom from C-1 of diol substrate giving a substrate radical that rearranges to a product radical by 1,2-shift of hydroxyl from C-2 to C-1. It has been proposed that the rearrangement mechanism involves the action of acidic and basic residues in the protein, with the involvement of a bridged intermediate. iv Dong Jiang, Newcastle University, PhD Thesis, 2010 Ethanolamine ammonia-lyase (EAL) from e.g. Clostridia sp. converts ethanolamine into acetaldehyde and ammonia by a similar pathway. Fluorine-substituted probes for coenzyme B12-dependent enzymatic reactions: 3,3,3- trifluoropropane-1,2-diol for diol dehydratase and 2-amino-3,3,3-trifluoropropanol for EAL, have been synthesised. 3,3,3-Trifluoropropane-1,2-diol is a substrate for glycerol dehydratase (ca. 4 % of the activity of propane-1,2-diol). This result is surprisingly in the context of the proposed mechanism for diol dehydratase, although ab initio molecular orbital calculations (collaboration with Dr D. M. Smith, Zagreb) have indicated that CF3 has a minimal effect on the stabilities of the proposed intermediates. A model study (performed in collaboration with Dr C. Chatgilialoglu, Bologna) showed that 1,1-difluoro-3-hydroxylpropanone was formed by continuous -irradiation of 3,3,3-trifluoropropane-1,2-diol. The expected 3,3,3-trifluoropropanal and 1,1,1-trifluoroacetone could not be determined from the 1H NMR analysis of the irradiation mixuture. Propanal and acetone were formed by the continuous - irradiation of propane-1,2-diol (analysis by GC and GC-MS, and by formation of the corresponding 2,4-dinitrophenylhydrazones). 2-Amino-3,3,3-trifluoropropanol was found to be devoid of any activity against EAL according to a coupled assay with alcohol dehydrogenase (collaboration with Dr G. H. Reed, University of Wisconsin-Madison). EPR study (collaboration with Dr K. Warncke, Emory University) has concluded that the CF3-substrate did not lead to the significant formation of a paramagnetic intermediate. This may be due to the CF3 group reducing the basicity of the amino group and the acidity of the alcohol group and so that there is a significant diminished interaction with the active site in EAL. Or this may be caused by the significant steric effect introduced by the trifluoromethyl group (CF3 group is between two and three times larger than CH3).