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 car bamylphosphate synthetase and aspartate transcarbamylase activities. G enetic and proteolysis studies suggested that the ATCase activity is c arried out by an independently folded domain. In order to provide stru ctural information for ongoing mutagenesis studies, a model of the thr ee-dimensional structure of this domain was generated on the basis of the known X-ray structure of the related catalytic subunit from E. col i ATCase. First, a model of the catalytic monomer was built and refine d by energy minimization. In this structure, the conserved residues be tween the two proteins were found to constitute the hydrophobic core w hereas almost all the mutated residues are located at the surface. The n, a trimeric structure was generated in order to build the active sit e as it lies at the interface between adjacent chains in the E. coli c atalytic trimer. After docking a bisubstrate analog into the active si te, the whole structure was energy minimized to regularize the interac tions at the contact areas between subunits. The resulting model is ve ry similar to that obtained for the E. coli catalytic trimer by X-ray crystallography, with a remarkable conservation of the structure of th e active site and its vicinity. Most of the interdomain and intersubun it interactions that are essential for the stability of the E. coli ca talytic 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 loo p which, in E. coli ATCase, is at the surface of the protein. This ins ertion exposes hydrophobic residues to the solvent. Interestingly, suc h an insertion is present in all the eukaryotic ATCase genes sequenced so far, suggesting that this region is interacting with another domai n of the multifunctional protein. (C) 1991 Wiley-Liss, Inc.