title: Evolutionarily distinct versions of the multidomain enzyme α-isopropylmalate synthase share discrete mechanisms of V-type allosteric regulation.
abstract: Understanding the evolution of allostery in multidomain enzymes remains an important step in improving our ability to identify and exploit structure-function relationships in allosteric mechanisms. A recent protein similarity network for the DRE-TIM metallolyase superfamily indicated there are two evolutionarily distinct forms of the enzyme α-isopropylmalate synthase (IPMS) sharing approximately 20% sequence identity. IPMS from Mycobacterium tuberculosis has been extensively characterized with respect to catalysis and the mechanism of feedback regulation by l-leucine. Here, IPMS from Methanococcus jannaschii (MjIPMS) is used as a representative of the second form of the enzyme, and its catalytic and regulatory mechanism is compared with that of MtIPMS to identify any functional differences between the two forms. MjIPMS exhibits kinetic parameters similar to those of other reported IPMS enzymes and is partially inhibited by l-leucine in a V-type manner. Identical values of (D2O)kcat (3.1) were determined in the presence and absence of l-leucine, indicating the hydrolytic step is rate-determining in the absence of l-leucine and remains so in the inhibited form of the enzyme. This mechanism is identical to the mechanism identified for MtIPMS ((D2O)kcat = 3.3 ± 0.3 in the presence of l-leucine) despite product release being rate-determining in the uninhibited MtIPMS enzyme. The identification of identical regulatory mechanisms in enzymes with low sequence identity raises important evolutionary questions concerning the acquisition and divergence of multidomain allosteric enzymes and highlights the need for caution when comparing regulatory mechanisms for homologous enzymes.
authors.affiliation: Department of Chemistry, The University of Alabama , Tuscaloosa, Alabama 35487, United States.
authors.forename: Garima
authors.initials: G
authors.lastname: Kumar
authors.forename: Patrick A
authors.initials: PA
authors.lastname: Frantom
mesh_terms: D015079:2-Isopropylmalate Synthase / Q000037:antagonists & inhibitors / Q000737:chemistry*; D000494:Allosteric Regulation; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry*; D055162:Biocatalysis; D019143:Evolution, Molecular; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007930:Leucine / Q000737:chemistry; D063747:Methanocaldococcus / Q000201:enzymology; D008958:Models, Molecular; D009169:Mycobacterium tuberculosis / Q000201:enzymology; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D013045:Species Specificity
chemical_list: D001426:Bacterial Proteins; D015079:2-Isopropylmalate Synthase; D007930:Leucine