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The glutamate mutase dependent on adenosylcobalamin (coenzyme B12) 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 epsilon 2 (epsilon, 53.5 kDa) and S, a monomer (sigma, 14.8 kDa). 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 B12. 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 B12 resulted in the formation of a fully active
glutamate mutase heterotetramer (epsilon 2 sigma 2) containing one molecule of
coenzyme B12. EPR spectra of recombinant glutamate mutase, now available in
sufficiency large amounts, were recorded after incubation of the enzyme with
coenzyme B12 and (S)-glutamate. The EPR signals (gx,y approximately 2.1, gz =
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(II)alamin (gx,y = 2.25). The spin concentration was 34-50% of the
concentration of the enzyme (epsilon 2 sigma 2) 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)-[2,3,3,4,4-2H5]glutamate induced a signal
significantly different to that of (S)-glutamate with an intensity of only 7%.
These data suggest an involvement of the Co(II)-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 predeuterated glutamate is
probably due to a kinetic isotope effect and indicates a cleavage of a C-H bond
during formation of the paramagnetic species.(ABSTRACT TRUNCATED AT 400 WORDS)
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