Kinetics and mechanism of the citrate synthase from the thermophilic archaeon Thermoplasma acidophilum

The kinetics and mechanism of the citrate synthase from a moderate thermoph ile, Thermoplasma acidophilum (TpCS), are compared with those of the citrat e synthase from a mesophile, pig heart (PCS). All discrete steps in the mec hanistic sequence of PCS can be identified in TpCS. The catalytic strategie s identified in PCS, destabilization of the oxaloacetate substrate carbonyl and stabilization of the reactive species, acetyl-CoA enolate, are present in TpCS. Conformational changes, which allow the enzyme to efficiently cat alyze both condensation of;acetyl-CoA thioester and subsequently hydrolysis of citryl-CoA thioester within the same active site, occur in both enzymes . However, significant differences exist between the two enzymes. PCS is a characteristically efficient enzyme: no internal step is clearly rate-limit ing and the condensation step is readily reversible. TpCS is a less efficie nt catalyst. Over a broad temperature range, inadequate stabilization of th e transition state for citryl-CoA hydrolysis renders this step nearly rate- limiting for the forward reaction of TpCS. Further, excessive stabilization of the citryl-CoA intermediate renders the condensation step nearly irreve rsible. Values of substrate and solvent deuterium isotope effects are consi stent with the kinetic model. Near its temperature optimum (70 degrees C), there is a modest increase in the reversibility of the condensation step fo r TpCS, but reversibility still falls short of that shown by PCS at 37 degr ees C. The root cause of the catalytic inefficiency of TpCS may lie in the lack of protein flexibility imposed by the requirement for thermal stabilit y of the protein itself or its temperature-labile substrate, oxaloacetate.