The Slow-Onset Nature of Allosteric Inhibition in α-Isopropylmalate Synthase from Mycobacterium tuberculosis Is Mediated by a Flexible Loop

The identification of structure-function relationships in allosteric enzymes is essential to describing a molecular mechanism for allosteric processes. The enzyme alpha-isopropylmalate synthase from Mycobacterium tuberculosis (MtIPMS) is subject to slow-onset, allosteric inhibition by L-leucine. Here we report that alternate amino acids act as rapid equilibrium noncompetitive inhibitors of MtIPMS failing to display biphasic inhibition kinetics. Amino acid substitutions on a flexible loop covering the regulatory binding pocket generate enzyme variants that have significant affinity for L-leucine but lack biphasic inhibition kinetics. Taken together, these results are consistent with the flexible loop mediating the slow-onset step of allosteric inhibition.