Abstract

The long range goal of this research program is to understand mechanisms of regulation in multisubunit proteins and other macromolecular assemblies. Our immediate goal is to elucidate the allosteric mechanism of E. coli aspartate transcarbamylase via analysis of the functional energetics of protein-protein and protein-ligand interactions and correlation of energetic and structural information. An analysis of the macroscopic thermodynamic properties of this system has been completed and atomic coordinates for three major conformational states are now available, making it possible to begin mapping pathways of energy transduction within the structure. Major goals of the next grant period will be to: (a) Continue the development of low and high pressure analytical gel chromatographic methods and apply them to the analysis of previously inaccessible protein- protein interactions in the catalytic subunit (c3), regulatory subunit (r2) and native enzyme (c6r6) over a range of conditions. (b) Extend the experimental analysis of electrostatic effects by (i) carrying out a comprehensive analysis of the effects of varying pH, ionic strength and specific ions on the structure, structural dynamics, energetics and function of wild type ATCase and its subunits, (ii)testing hypotheses developed by computer modelling in the previous grant period by examining the structure, energetics and functional properties of single site mutants in which groups calculated to undergo large pK changes upon assembly or ligand binding are modified, (iii) investigating the mechanism of synergistic inhibition by CTP and UTP, particularly the role of ionizable groups, by site-directed mutagenesis and enzymatic assay. (c) Develop cryoisoelectric focussing as a method for analyzing populations of ligated species and analyze the populations of PALA-ligated species over a range of pH and temperature and in the presence and absence of CTP and ATP. (d) Develop statistical thermodynamic models of the allosteric mechanism.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37DK017335-20
Application #
3483194
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Project Start
1991-09-30
Project End
1994-12-31
Budget Start
1992-02-01
Budget End
1992-12-31
Support Year
20
Fiscal Year
1992
Total Cost
Indirect Cost

  Institution

Name
University of Minnesota Twin Cities
Department
Type
Schools of Medicine
DUNS #
168559177
City
Minneapolis
State
MN
Country
United States
Zip Code
55455

  Publications

LiCata, V J; Burz, D S; Moerke, N J et al. (1998) The magnitude of the allosteric conformational transition of aspartate transcarbamylase is altered by mutations. Biochemistry 37:17381-5
Hariharan, M; Allewell, N M (1998) Effects of the T-->R transition on the electrostatic properties of E. coli aspartate transcarbamylase. Proteins 32:200-10
LiCata, V J; Allewell, N M (1998) Solvent perturbation of the allosteric regulation of aspartate transcarbamylase. Biochim Biophys Acta 1384:306-14
Ha, Y; Allewell, N M (1998) Intersubunit hydrogen bond acts as a global molecular switch in Escherichia coli aspartate transcarbamoylase. Proteins 33:430-43
LiCata, V J; Allewell, N M (1997) Is substrate inhibition a consequence of allostery in aspartate transcarbamylase? Biophys Chem 64:225-34
Morizono, H; Tuchman, M; Rajagopal, B S et al. (1997) Expression, purification and kinetic characterization of wild-type human ornithine transcarbamylase and a recurrent mutant that produces 'late onset' hyperammonaemia. Biochem J 322 ( Pt 2):625-31
LiCata, V J; Allewell, N M (1997) Functionally linked hydration changes in Escherichia coli aspartate transcarbamylase and its catalytic subunit. Biochemistry 36:10161-7
Morizono, H; Listrom, C D; Rajagopal, B S et al. (1997) 'Late onset' ornithine transcarbamylase deficiency: function of three purified recombinant mutant enzymes. Hum Mol Genet 6:963-8
Oberoi, H; Trikha, J; Yuan, X et al. (1996) Identification and analysis of long-range electrostatic effects in proteins by computer modeling:aspartate transcarbamylase. Proteins 25:300-14
Yuan, X; LiCata, V J; Allewell, N M (1996) Effects of assembly and mutations outside the active site on the functional pH dependence of Escherichia coli aspartate transcarbamylase. J Biol Chem 271:1285-94

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