title: Structural properties of UMP-kinase from Escherichia coli: modulation of protein solubility by pH and UTP.
abstract: UMP-kinase from Escherichia coli, unlike the analogous enzyme from eukaryotic organisms, is an oligomeric protein subjected to complex regulatory mechanisms in which UTP and GTP act as allosteric effectors. While the enzyme has an unusually low solubility at neutral pH (< or = 0.1 mg of protein/ mL), its solubility increases markedly above pH 8 and below pH 4. Furthermore, the solubility of the bacterial UMP-kinase at neutral pH is greatly enhanced in the presence of Mg-free UTP. Thermal denaturation experiments have demonstrated that UTP also increases the stability of the protein. Fourier-transform infrared spectroscopy and circular dichroism show that the secondary structure of the protein is the same at neutral and at alkaline pH. These data indicate that variations in enzyme solubility must be related to subtle changes in the tertiary and/or quaternary structure which modulate the exposure of hydrophobic surfaces in the protein molecule. A variant of UMP-kinase, obtained by site-directed mutagenesis (Asp159Asn), which is similar to the wild-type enzyme in its stability and kinetic properties, has a much increased water solubility (> 5 mg protein/mL) even at neutral pH. This suggests that salt bridges may be involved in the equilibrium between the soluble and aggregated forms of the wild-type enzyme, and that conformational changes induced upon binding of UTP increase the protein solubility by disrupting these salt bridges.
authors.affiliation: Unité de Biochimie des Régulations Cellulaires, Institut Pasteur, Paris, France.
authors.forename: L
authors.initials: L
authors.lastname: Serina
authors.forename: N
authors.initials: N
authors.lastname: Bucurenci
authors.forename: A M
authors.initials: AM
authors.lastname: Gilles
authors.forename: W K
authors.initials: WK
authors.lastname: Surewicz
authors.forename: H
authors.initials: H
authors.lastname: Fabian
authors.forename: H H
authors.initials: HH
authors.lastname: Mantsch
authors.forename: M
authors.initials: M
authors.lastname: Takahashi
authors.forename: I
authors.initials: I
authors.lastname: Petrescu
authors.forename: G
authors.initials: G
authors.lastname: Batelier
authors.forename: O
authors.initials: O
authors.lastname: Bârzu
mesh_terms: D000494:Allosteric Regulation / Q000187:drug effects; D001483:Base Sequence; D002942:Circular Dichroism; D017931:DNA Primers / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009703:Nucleoside-Phosphate Kinase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D009711:Nucleotides / Q000494:pharmacology; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012995:Solubility / Q000187:drug effects; D013050:Spectrometry, Fluorescence; D017550:Spectroscopy, Fourier Transform Infrared; D014461:Ultracentrifugation; D014544:Uridine Triphosphate / Q000378:metabolism / Q000494:pharmacology
chemical_list: D017931:DNA Primers; D009711:Nucleotides; C023276:uridine monophosphate kinase; D009703:Nucleoside-Phosphate Kinase; D014544:Uridine Triphosphate