title: Glutamate 636 of the Escherichia coli pyruvate dehydrogenase-E1 participates in active center communication and behaves as an engineered acetolactate synthase with unusual stereoselectivity.
abstract: The residue Glu636 is located near the thiamine diphosphate (ThDP) binding site of the Escherichia coli pyruvate dehydrogenase complex E1 subunit (PDHc-E1), and to probe its function two variants, E636A and E636Q were created with specific activities of 2.5 and 26% compared with parental PDHc-E1. According to both fluorescence binding and kinetic assays, the E636A variant behaved according to half-of-the-sites mechanism with respect to ThDP. In contrast, with the E636Q variant a K(d,ThDP) = 4.34 microM and K(m,ThDP) = 11 microM were obtained with behavior more reminiscent of the parental enzyme. The CD spectra of both variants gave evidence for formation of the 1',4'-iminopyrimidine tautomer on binding of phosphonolactylthiamine diphosphate, a stable analog of the substrate-ThDP covalent complex. Rapid formation of optically active (R)-acetolactate by both variants, but not by the parental enzyme, was observed by CD and NMR spectroscopy. The acetolactate configuration produced by the Glu636 variants is opposite that produced by the enzyme acetolactate synthase and the Asp28-substituted variants of yeast pyruvate decarboxylase, suggesting that the active centers of the two sets of enzymes exhibit different facial selectivity (re or si) vis à vis pyruvate. The tryptic peptide map (mass spectral analysis) revealed that the Glu636 substitution changed the mobility of a loop comprising amino acid residues from the ThDP binding fold. Apparently, the residue Glu636 has important functions both in active center communication and in protecting the active center from undesirable "carboligase" side reactions.
authors.affiliation: Department of Chemistry, Rutgers University, Newark, New Jersey 07102, USA. nemeria@andromeda.rutgers.edu
authors.forename: Natalia
authors.initials: N
authors.lastname: Nemeria
authors.forename: Kai
authors.initials: K
authors.lastname: Tittmann
authors.forename: Ebenezer
authors.initials: E
authors.lastname: Joseph
authors.forename: Leon
authors.initials: L
authors.lastname: Zhou
authors.forename: Michelle B
authors.initials: MB
authors.lastname: Vazquez-Coll
authors.forename: Palaniappa
authors.initials: P
authors.lastname: Arjunan
authors.forename: Gerhard
authors.initials: G
authors.lastname: Hübner
authors.forename: William
authors.initials: W
authors.lastname: Furey
authors.forename: Frank
authors.initials: F
authors.lastname: Jordan
mesh_terms: D000095:Acetolactate Synthase / Q000737:chemistry / Q000502:physiology*; D001224:Aspartic Acid / Q000737:chemistry; D001665:Binding Sites; D002384:Catalysis; D002942:Circular Dichroism; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000201:enzymology*; D014644:Genetic Variation; D018698:Glutamic Acid / Q000737:chemistry*; D007700:Kinetics; D007773:Lactates / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008956:Models, Chemical; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011741:Pyrimidine Nucleosides / Q000737:chemistry; D030481:Pyruvate Dehydrogenase (Lipoamide) / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D017550:Spectroscopy, Fourier Transform Infrared; D013237:Stereoisomerism; D013696:Temperature; D013835:Thiamine Pyrophosphate / Q000737:chemistry; D014357:Trypsin / Q000494:pharmacology; D014466:Ultraviolet Rays
chemical_list: D007773:Lactates; D010455:Peptides; D011741:Pyrimidine Nucleosides; D001224:Aspartic Acid; D018698:Glutamic Acid; D030481:Pyruvate Dehydrogenase (Lipoamide); D000095:Acetolactate Synthase; D014357:Trypsin; D013835:Thiamine Pyrophosphate; D010100:Oxygen