Kinetics and product analysis of the reaction catalysed by recombinant homoaconitase from Thermus thermophilus

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 α-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 (2 R ,3 S )-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 (2 R ,3 S )-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 α-aminoadipate pathway can accept analogous tricarboxylic acid-cycle substrates. The enzyme shows no apparent feedback inhibition by L-lysine.