Purification and Characteristics of Aspartate Transcarbamylase from Pseudomonas fluorescens

Aspartate transcarbamylase from a Pseudomonas fluerescens has been purified to homogeneity.The enzyme has a molecular weight of 360,000.It is composed of 2 apparently identical subunits with molecular weights of 180,000.Activity can be recovered from the enzyme denatured with mercaptoethanol and sodium dodecyl sulfate when the dodecyl sulfate is dialyzed away.The renatured enzyme has the molecular weight of the native enzyme.Carbamyl phosphate synthetase activity is lost continuously during purification, so that it is unlikely that this bacterium possesses a carbamyl phosphate synthetase-aspartate transcarbamylase complex such as is found in Neurospora and yeast.The kinetic and regulatory properties of aspartate transcarbamylase from P. fluorescens were also studied.The substrate saturation curves in the absence and presence of all inhibitors tested are hyperbolic.With limiting carbamyl phosphate, all trinucleotides tested, and CDP, pyrophosphate, and orthophosphate inhibit competitively at micromolar concentrations.When carbamyl phosphate is saturating a second binding site for the nucleotides is evident, but pyrophosphate and orthophosphate no longer inhibit.At millimolar concentrations, the nucleotides are noncompetitive inhibitors with aspartate.The function of this regulatory site cannot be separated from the catalytic site by mild heat treatment.In bacteria, the pyrimidine biosynthetic pathway is controlled in part by feedback inhibition or activation of one or both of the first two enzymes of the pathway, i.e. carbamyl phosphate synthetase or aspartate transcarbamylase (l-7).ATCase' usually