title: Kinetic analysis of β-galactosidase and β-glucuronidase tetramerization coupled with protein translation.
abstract: Both β-galactosidase (GAL) and β-glucuronidase (GUS) are tetrameric enzymes used widely as reporter proteins. However, little is known about the folding and assembly of these enzymes. Although the refolding kinetics of GAL from a denatured enzyme have been reported, it is not known how the kinetics differ when coupled with a protein translation reaction. Elucidating the assembly kinetics of GAL and GUS when coupled with protein translation will illustrate the differences between these two reporter proteins and also the assembly process under conditions more relevant to those in vivo. In this study, we used an in vitro translation/transcription system to synthesize GAL and GUS, measured the time development of the activity and oligomerization state of these enzymes, and determined the rate constants of the monomer to tetramer assembly process. We found that at similar concentrations, GAL assembles into tetramers faster than GUS. The rate constant of monomer to dimer assembly of GAL was 50-fold faster when coupled with protein translation than that of refolding from the denatured state. Furthermore, GAL synthesis was found to lack the rate-limiting step in the assembly process, whereas GUS has two rate-limiting steps: monomer to dimer assembly and dimer to tetramer assembly. The consequence of these differences when used as reporter proteins is discussed.
authors.affiliation: Department of Bioinformatic Engineering, Graduate School of Information Science and Technology, Osaka University, Osaka, Japan.
authors.forename: Tomoaki
authors.initials: T
authors.lastname: Matsuura
authors.forename: Kazufumi
authors.initials: K
authors.lastname: Hosoda
authors.forename: Norikazu
authors.initials: N
authors.lastname: Ichihashi
authors.forename: Yasuaki
authors.initials: Y
authors.lastname: Kazuta
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Yomo
mesh_terms: D057075:Enzyme Assays; D007700:Kinetics; D014176:Protein Biosynthesis; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D014158:Transcription, Genetic; D001616:beta-Galactosidase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001617:beta-Glucosidase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D001617:beta-Glucosidase; D001616:beta-Galactosidase