title: Use of analytical gel chromatography to analyze tertiary and quaternary structural changes in E. coli aspartate transcarbamylase.
abstract: E. coli aspartate transcarbamylase (ATCase) is a large (310 kDa) protein that undergoes major changes in quaternary structure when substrates and regulatory nucleotides bind. We have used analytical gel chromatography to detect quaternary structure changes in both the holoenzyme and its catalytic subunit (c3), to characterize the quaternary structure of single site mutant proteins and to monitor urea-induced dissociation and unfolding of c3. Binding of the bisubstrate analog PALA (N-(phosphonacetyl)-L-aspartate) to ATCase and c3 has been shown to alter s20.w by -3.3% and + 1.4%, respectively [Howlett, G.J. and Schachman, H.K. (1977), Biochemistry 23, 5077-5083]. The corresponding changes in the chromatographic partition coefficient (sigma) are -2.6 +/- 0.3% and 5.5 +/- 1.9% on Sephacryl S400HR and S200, respectively. Partition coefficients of mutant ATCases with single site mutations in the c chain differ from those of the wild-type protein by +/- 0.5% in small zone experiments; for example, mutations Arg 269----Gly and Glu 239----Gln alter the partition coefficient by 0.4% and -0.5%, respectively. The partition coefficient of mutant Glu 50----Gln is identical to the wild type enzyme. In the presence of saturating PALA, partition coefficients of Glu 50----Gln and Arg 269----Gly, but not Glu 239----Gln are identical to those of the wild type. Results for Glu 239----Gln are consistent with measurements of activity, small angle X-ray scattering and sedimentation coefficient that indicate that mutations at this site shift the quaternary structure towards the R state [Ladjimi and Kantrowitz (1988), Biochemistry 27, 276-83; Vachette and Hervé, cited by Kantrowitz and Lipscomb (1988), Science 241, 669-674; Newell and Schachman (1988), FASEB J. 2, A551]. Results for Glu 50----Gln are also consistent with measurements of activity (Ladjimi et al. (1988), Biochemistry 27, 268-276). The changes in tertiary and quaternary structure that result from urea-induced denaturation of c3 result in larger changes in the partition coefficient. Dissociation into folded monomers in 1-1.75 M urea is accompanied by a 4.6% increase in partition coefficient, while denaturation at greater than 5 M urea gives rise to a 43% decrease on S-300 Sephacryl. The bisubstrate analog PALA suppresses dissociation and increases the cooperativity of the unfolding reaction.
authors.affiliation: Department of Molecular Biology and Biochemistry, Wesleyan University, Middletown, CT 06457.
authors.forename: S
authors.initials: S
authors.lastname: Bromberg
authors.forename: D S
authors.initials: DS
authors.lastname: Burz
authors.forename: N M
authors.initials: NM
authors.lastname: Allewell
mesh_terms: D001221:Aspartate Carbamoyltransferase / Q000235:genetics / Q000378:metabolism; D002850:Chromatography, Gel; D004926:Escherichia coli / Q000201:enzymology*; D008024:Ligands; D046911:Macromolecular Substances; D009154:Mutation; D011487:Protein Conformation; D011489:Protein Denaturation; D013329:Structure-Activity Relationship; D014508:Urea
chemical_list: D008024:Ligands; D046911:Macromolecular Substances; D014508:Urea; D001221:Aspartate Carbamoyltransferase