Kinetic mechanism of uracil phosphoribosyltransferase from Escherichia coli and catalytic importance of the conserved proline in the PRPP binding site.

Phosphoribosyltransferases catalyze the formation of nucleotides from a nitrogenous base and 5-phosphoribosyl-alpha-1-pyrophosphate (PRPP). These enzymes and the PRPP synthases resemble each other in a short homologous sequence of 13 amino acid residues which has been termed the PRPP binding site and which interacts with the ribose 5-phosphate moiety in structurally characterized complexes of PRPP and nucleotides. We show that each class of phosphoribosyltransferases has subtle deviations from the general consensus PRPP binding site and that all uracil phosphoribosyltransferases (UPRTases) have a proline residue at a position where other phosphoribosyltransferases and the PRPP synthases have aspartate. To investigate the role of this unusual proline (Pro 131 in the E. coli UPRTase) for enzyme activity, we changed the residue to an aspartate and purified the mutant P131D enzyme to compare its catalytic properties with the properties of the wild-type protein. We found that UPRTase of E. coli obeyed the kinetics of a sequential mechanism with the binding of PRPP preceding the binding of uracil. The basic kinetic constants were derived from initial velocity measurements, product inhibition, and ligand binding assays. The change of Pro 131 to Asp caused a 50-60-fold reduction of the catalytic rate (kcat) in both directions of the reaction and approximately a 100-fold increase in the KM for uracil. The KM for PRPP was strongly diminished by the mutation, but kcat/KM,PRPP and the dissociation constant (KD,PRPP) were nearly unaffected. We conclude that the proline in the PRPP binding site of UPRTase is of only little importance for binding of PRPP to the free enzyme, but is critical for binding of uracil to the enzyme-PRPP complex and for the catalytic rate.