HACN (
homoaconitase) is a member of a family of [4Fe-4S] cluster-dependent
enzymes that catalyse hydration/
dehydration reactions. The best characterized example of this family is the ubiquitous
ACN (
aconitase), which catalyses the
dehydration of
citrate to cis-
aconitate, and the subsequent hydration of cis-
aconitate to
isocitrate. HACN is an
enzyme from the alpha-aminoadipate pathway of
lysine biosynthesis, and has been identified in higher fungi and several archaea and one thermophilic species of bacteria, Thermus thermophilus. HACN catalyses the hydration of cis-homoaconitate to (2R,3S)-homoisocitrate, but the HACN-catalysed
dehydration of (R)-
homocitrate to cis-homoaconitate has not been observed in vitro. We have synthesized the substrates and putative substrates for this
enzyme, and in the present study report the first steady-state kinetic data for recombinant HACN from T. thermophilus using a (2R,3S)-homoisocitrate
dehydrogenase-coupled assay. We have also examined the products of the reaction using HPLC. We do not observe HACN-catalysed '
homocitrate dehydratase' activity; however, we have observed that
ACN can catalyse the
dehydration of (R)-
homocitrate to cis-homoaconitate, but HACN is required for subsequent conversion of cis-homoaconitate into
homoisocitrate. This suggests that the in vivo process for conversion of
homocitrate into
homoisocitrate requires two
enzymes, in simile with the
propionate utilization pathway from Escherichia coli. Surprisingly, HACN does not show any activity when cis-
aconitate is substituted for the substrate, even though other
enzymes from the alpha-aminoadipate pathway can accept analogous tricarboxylic acid-cycle substrates. The
enzyme shows no apparent feedback inhibition by
L-lysine.