title: Kinetic evidence for interdomain communication in the allosteric regulation of alpha-isopropylmalate synthase from Mycobacterium tuberculosis.
abstract: The enzyme alpha-isopropylmalate synthase from Mycobacterium tuberculosis (MtIPMS) has been identified as a possible target for the design of new antitubercular therapeutics. Recently, it was shown that MtIPMS is subject to slow-onset, feedback inhibition by l-leucine, the first instance of an allosteric regulator utilizing this mechanism. Structural studies are inconsistent with canonical allosteric mechanisms, including changes to the quaternary structure or large, rigid-body conformational changes to the enzyme upon l-leucine binding. Thus, the allosteric regulation may result from a discrete inhibitory signal transmitted to the active site upon l-leucine binding in the regulatory domain, a distance of more than 50 A. To test this mechanism, site-directed mutagenesis was employed to construct enzymes with substitutions at phylogenetically conserved active site residues near the interface of the catalytic and linker domains. The substitutions had wide-ranging effects on the kinetics of l-leucine inhibition, with some modest effects on the kinetic parameters of catalysis. The most dramatic result was the finding that the Y410F mutant form of MtIPMS is insensitive to l-leucine inhibition, suggesting that this residue has completely uncoupled the inhibitory signal to the active site. Overall, the data are consistent with a mechanism of allosteric regulation described by the interdomain communication of the inhibitory signal from the regulatory to catalytic domain and implicate the interactions between the linker and catalytic domains as critical determinants of inhibitory signal transmission.
authors.affiliation: Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA.
authors.forename: Luiz Pedro S
authors.initials: LP
authors.lastname: de Carvalho
authors.forename: Patrick A
authors.initials: PA
authors.lastname: Frantom
authors.forename: Argyrides
authors.initials: A
authors.lastname: Argyrou
authors.forename: John S
authors.initials: JS
authors.lastname: Blanchard
mesh_terms: D015079:2-Isopropylmalate Synthase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000494:Allosteric Regulation; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D002151:Calorimetry / Q000379:methods; D002384:Catalysis / Q000187:drug effects; D007700:Kinetics; D007930:Leucine / Q000378:metabolism / Q000494:pharmacology; D009682:Magnetic Resonance Spectroscopy; D008293:Malates / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D017186:Titrimetry
chemical_list: D001426:Bacterial Proteins; D008293:Malates; C005906:alpha-isopropylmalate; D015079:2-Isopropylmalate Synthase; D007930:Leucine