Mapping of the Allosteric Network in the Regulation of α-Isopropylmalate Synthase from Mycobacterium tuberculosis by the Feedback Inhibitor l-Leucine: Solution-Phase H/D Exchange Monitored by FT-ICR Mass Spectrometry

摘要

As it is becoming accepted that allosteric regulation can occur through a change in localnconformational equilibria as opposed to a change in overall static structure, a thorough description of thenstructural aspects of these types of mechanisms will be essential to understanding this fundamental biologicalnprocess. Here we report the experimental identification of key regions of conformational perturbation in thenallosteric network of a large (144 kDa), multidomain enzyme by use of solution-phase hydrogen/deuteriumnexchange. Large perturbations in the regulatory domain induced by effector molecule binding are linked to anvery specific, targeted perturbation in the active site, some 50 A ° away. Binding of L-leucine to an enzymenvariant (Y410F) that is kinetically insensitive to effector binding was shown to elicit similar changes in thenregulatory domain, but perturbs an alternate region of the catalytic domain, consistent with the proposednallosteric mechanism. These results comprise one of the first reports of an experimentally mapped allostericnmechanism in a protein of this size and provide necessary information to be used toward the development ofnallostery-based drugs or enzymes with engineered regulatory properties.