The glutamate mutase dependent on adenosylcobalamin (coenzyme B,J catalyzes the carbon skeleton rearrangement of (S)-glutamate to (2S, 3S)-3-methylaspartate, the first step of the glutamate fermentation pathway of the anaerobic bacterium Clostridium cochlearium. The enzyme consists of two protein components, E, a dimer E~ ( E , 53.5 kDa) and S, a monomer (a, 14.8 ma). The corresponding genes (glmE and glmS) were cloned, sequenced and over-expressed in Escherichia coli. The genes glmS and glmE are separated by glmL encoding a protein of unknown function. The deduced amino acid sequence of GlmL contains an ATP-binding motif which is common to chaperones of the HSP70-type, actin and procaryotic cell-cycle proteins.Both components of glutamate mutase were purified with excellent yields from cell-free extracts of E. coli carrying the corresponding genes. In contrast to component E, component S was shown to bind coenzyme BIZ. This observation strongly supports the idea that significant similarities of the amino acid sequences of component S and several other cobamide-dependent enzymes represent a common binding motif. Incubation of pure components E and S with coenzyme B,, resulted in the formation of a fully active glutamate mutase heterotetramer (~~a~) containing one molecule of coenzyme BIZ.EPR spectra of recombinant glutamate mutase, now available in sufficiently large amounts, were recorded after incubation of the enzyme with coenzyme B,, and (S)-glutamate. The EPR signals (gx, = 2.1, g, = 1.985) were of much better resolution than observed earlier with the clostridial enzyme. Their typical hyperfine splitting is clearly derived from Co(II), which is involved in the formation of the paramagnetic species but is different from cob(I1)alamin (gx,, = 2.25). The spin concentration was 34-50% of the concentration of the enzyme ( E~C T J coenzyme complex. The competitive inhibitors (2S, 4S)-4-fluoroglutamate and 2-methyleneglutarate induced similar but not identical signals with spin concentrations of 134-148% of the enzyme concentration. Even (S)- [2,3,3,4,]glutaate induced a signal significantly different to that of (S)-glutamate with an intensity of only 7 %. These data suggest an involvement of the Co(I1)-containing paramagnetic species in catalysis, the concentration of which reflects a steady state between its formation and decomposition. The large difference in the spin concentrations observed with (S)-glutamate as compared to the perdeuterated glutamate is probably due to a kinetic isotope effect and indicates a cleavage of a C-H bond during formation of the paramagnetic species. It is discussed that the paramagnetic species represents a radical pair composed of Co(I1) and an organic radical.Overnight incubation of glutamate mutase and coenzyme with substrate or one of the competitive inhibitors resulted in an inactive enzyme and cob(II)alamin, which was identified by EPR spectroscopy (gX,, = 2.25). This cob(I1)alamin appeared to be very similar to a cob(I1)amide present in inactive glutamate mutase preparations...

Both components, E and S, of the adenosylcobalamin-(coenzyme B1 ,)-dependent glutamate mutase from Clostridium cochlearium were purified. Component S (16 kDa) must be added to component E to obtain activity, although the latter contains substoichiometric amounts of component S besides the major 50-kDa subunit. The enzyme proved to be very similar to that of C . tetanomorphum Substitution of the migrating hydrogen at C-4 of glutamate by fluorine yielded the potent competitive inhibitor (2S,4S)-4-fluoroglutamate (Ki = 70 pM). (2R,3RS)-3-Fluoroglutamate (KI = 600 pM) was also inhibitory. The competitive inhibition by 2-methyleneglutarate (Ki = 400 pM) and (59-3-methylitaconate (Ki = 100 pM) but not by (RS)-2-methylglutarate suggested the transient formation of an sp2 center during catalysis. However, the presence of an N-terminal pyruvoyl residue was excluded and no evidence for the participation of another electrophilic center in the reaction was obtained.Glutamate mutase catalyses the reversible rearrangement of (S)-glutamate to (2S,3S)-3-methylaspartate, the first step in the fermentation of glutamate by Clostridium tetanomorphum [l] and related clostridia [2]. The elucidation of the coenzyme of this reaction as an adenosyl derivative of pseudovitamin B was a landmark in biochemistry [3] leading to the discovery of the family of coenzyme-B, 2-dependent enzymes (for reviews see 14-91). Until now, the role of coenzyme B12 in those rearrangements has not been clear.In general, homolytic cleavage of the cobalt-carbon bond is agreed to be the first step in the catalysis of adenosylcobalamin-dependent enzymes. The resulting S-deoxyadenosyl radical should abstract a hydrogen atom yielding the substrate radical which rearranges to the product radical. Redonation of a hydrogen leads to the product and regenerates the . Such a mechanism is unlikely in the glutamate mutase reaction due to the lack of a double bond at the migrating carbon. Indeed, chemical modelling showed that glutamate derivatives only rearranged if the amino acid was converted to a ketimine [16] suggesting the participation of an electrophilic center in the reaction. Pyridoxal phosphate, however, the most likely prosthetic group, was not detected [17].Glutamate mutase from C. tetanomorphum is composcd of two components: E (128 kDa) and S (17 kDa). Due to the instability of component E, only component S was purified to homogeneity [7, 181. This paper describes the purification of components E and S of the closely related C. cochlarium to high purity and investigations on the mechanistic questions

Highly active and cobamide-free ~lutamate mutase was obtained from ClostrMimn cochlearium by a modification oi" the original purification procedure. After incubation of the enzyme with dithiothreitol, adenosylcobalamin (coenzyme B~,) and the substrate (5)-glutamat¢, a paramaBnetic species was observed by EPR-speetroscopy. The signal was maximal within 15 ms after mi~in~ with ~lutamate. Different signals were detected after incubating the system with the competitive inhibitors (2S,4S)-4-flaoroglutamate or 2-methyleneglutarate instead of tbe substrate. The former developed with an at least 100-fold lower rate then the signal with glutamate. All three signals are probably due to low-spin ¢ob(ll)amide species with an extraordinary low g,.,. value as compared with cob(ll)alamin.