Mapping of the allosteric network in the regulation of alpha-isopropylmalate synthase from Mycobacterium tuberculosis by the feedback inhibitor L-leucine: solution-phase H/D exchange monitored by FT-ICR mass spectrometry.
As it is becoming accepted that allosteric regulation can occur through a change in local conformational equilibria as opposed to a change in overall static structure, a thorough description of the structural aspects of these types of mechanisms will be essential to understanding this fundamental biological process. Here we report the experimental identification of key regions of conformational perturbation in the allosteric network of a large (144 kDa), multidomain enzyme by use of solution-phase hydrogen/deuterium exchange. Large perturbations in the regulatory domain induced by effector molecule binding are linked to a very specific, targeted perturbation in the active site, some 50 A away. Binding of L-leucine to an enzyme variant (Y410F) that is kinetically insensitive to effector binding was shown to elicit similar changes in the regulatory domain, but perturbs an alternate region of the catalytic domain, consistent with the proposed allosteric mechanism. These results comprise one of the first reports of an experimentally mapped allosteric mechanism in a protein of this size and provide necessary information to be used toward the development of allostery-based drugs or enzymes with engineered regulatory properties.
Frantom
Patrick A
PA
Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Zhang
Hui-Min
HM
Emmett
Mark R
MR
Marshall
Alan G
AG
Blanchard
John S
JS
D015079:2-Isopropylmalate Synthase / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000378:metabolism; D000494:Allosteric Regulation; D041961:Deuterium Exchange Measurement; D015536:Down-Regulation; D025461:Feedback, Physiological; D007930:Leucine / Q000737:chemistry*; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D044367:Phase Transition; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012996:Solutions; D017550:Spectroscopy, Fourier Transform Infrared
D012996:Solutions; D015079:2-Isopropylmalate Synthase; D007930:Leucine