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

Patrick A. Frantom, Hui Min Zhang, Mark Emmett, Alan G. Marshall, John S. Blanchard

Research output: Contribution to journalArticle

32 Citations (Scopus)

Abstract

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 Å 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.

Original languageEnglish (US)
Pages (from-to)7457-7464
Number of pages8
JournalBiochemistry
Volume48
Issue number31
DOIs
StatePublished - Aug 11 2009
Externally publishedYes

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Mycobacterium tuberculosis
Leucine
Mass spectrometry
Mass Spectrometry
Ion exchange
Feedback
Catalytic Domain
Enzymes
Allosteric Regulation
Biological Phenomena
Deuterium
Hydrogen
Molecules
Pharmaceutical Preparations
Proteins

ASJC Scopus subject areas

  • Biochemistry

Cite this

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. / Frantom, Patrick A.; Zhang, Hui Min; Emmett, Mark; Marshall, Alan G.; Blanchard, John S.

In: Biochemistry, Vol. 48, No. 31, 11.08.2009, p. 7457-7464.

Research output: Contribution to journalArticle

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