Long-range allosteric transitions in carbamoyl phosphate synthetase

Area of Science:

• Biochemistry
• Structural Biology
• Enzyme Kinetics

Background:

• Carbamoyl phosphate synthetase (CPS) is crucial for pyrimidine and arginine biosynthesis.
• CPS from Escherichia coli has distinct small and large subunits with three active sites linked by a tunnel.
• Allosteric regulation by compounds like UMP, IMP, and L-ornithine is known.

Purpose of the Study:

• To determine the three-dimensional structure of a site-directed mutant of E. coli carbamoyl phosphate synthetase (C248D).
• To investigate the structural basis for altered glutaminase activity and abolished carbamoyl phosphate synthesis in the mutant.
• To elucidate the binding mode of urate monophosphate (UMP) in the mutant enzyme.

Main Methods:

• Site-directed mutagenesis of carbamoyl phosphate synthetase (Cys 248 to Asp).
• X-ray crystallography to determine the three-dimensional structure of the mutant protein.
• Analysis of electron density maps to identify bound ligands.

Main Results:

• The C248D mutant exhibited a 40-fold increase in glutaminase activity but abolished carbamoyl phosphate synthesis.
• Despite not being added, urate monophosphate (UMP) was unexpectedly found bound to the enzyme.
• The structure revealed the binding site and manner of UMP interaction within the mutant carbamoyl phosphate synthetase.

Conclusions:

• The C248D mutation significantly alters the catalytic and regulatory properties of carbamoyl phosphate synthetase.
• The unexpected UMP binding provides novel structural information on enzyme-ligand interactions.
• This study enhances understanding of carbamoyl phosphate synthetase structure-function relationships and allosteric regulation.