Structural modeling and electrostatic properties of aspartate transcarbamylase from Saccharomyces cerevisiae.
In Saccharomyces cerevisiae the first two reactions of the pyrimidine pathway are catalyzed by a multifunctional protein which possesses carbamylphosphate synthetase and aspartate transcarbamylase activities. Genetic and proteolysis studies suggested that the ATCase activity is carried out by an independently folded domain. In order to provide structural information for ongoing mutagenesis studies, a model of the three-dimensional structure of this domain was generated on the basis of the known X-ray structure of the related catalytic subunit from E. coli ATCase. First, a model of the catalytic monomer was built and refined by energy minimization. In this structure, the conserved residues between the two proteins were found to constitute the hydrophobic core whereas almost all the mutated residues are located at the surface. Then, a trimeric structure was generated in order to build the active site as it lies at the interface between adjacent chains in the E. coli catalytic trimer. After docking a bisubstrate analog into the active site, the whole structure was energy minimized to regularize the interactions at the contact areas between subunits. The resulting model is very similar to that obtained for the E. coli catalytic trimer by X-ray crystallography, with a remarkable conservation of the structure of the active site and its vicinity. Most of the interdomain and intersubunit interactions that are essential for the stability of the E. coli catalytic trimer are maintained in the yeast enzyme even though there is only 42% identity between the two sequences. Free energy calculations indicate that the trimeric assembly is more stable than the monomeric form. Moreover an insertion of four amino acids is localized in a loop which, in E. coli ATCase, is at the surface of the protein. This insertion exposes hydrophobic residues to the solvent. Interestingly, such an insertion is present in all the eukaryotic ATCase genes sequences so far, suggesting that this region is interacting with another domain of the multifunctional protein.
Villoutreix
B O
BO
Laboratoire d'Enzymologie, CNRS, 91198, Gif-sur-Yvette, France.
Spassov
V Z
VZ
Atanasov
B P
BP
Hervé
G
G
Ladjimi
M M
MM
D000595:Amino Acid Sequence; D001221:Aspartate Carbamoyltransferase / Q000737:chemistry* / Q000378:metabolism; D001224:Aspartic Acid / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D003198:Computer Simulation; D004560:Electricity; D004926:Escherichia coli / Q000201:enzymology; D005069:Evaluation Studies as Topic; D007477:Ions; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010746:Phosphonoacetic Acid / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D007477:Ions; D001224:Aspartic Acid; C013195:sparfosic acid; D001221:Aspartate Carbamoyltransferase; D010746:Phosphonoacetic Acid