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 nit rogenous base and 5-phosphoribosyl-alpha-1-pyrophosphate (PRPP). These enzy mes and the PRPP synthases resemble each other in a short homologous sequen ce of 13 amino acid residues which has been termed the PRPP binding site an d which interacts with the ribose 5-phosphate moiety in structurally charac terized complexes of PRPP and nucleotides. We show that each class of phosp horibosyltransferases has subtle deviations from the general consensus PRPP binding site and that all uracil phosphoribosyltransferases (UPRTases) hav e a proline residue at a position where other phosphoribosyltransferases an d the PRPP synthases have aspartate. To investigate the role of this unusua l 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 kineti c constants were derived from initial velocity measurements, product inhibi tion, and ligand binding assays. The change of Pro 131 to Asp caused a 50-6 0-fold reduction of the catalytic rate (k(cat)) in both directions of the r eaction and approximately a 100-fold increase in the K-M for uracil. The K- M for PRPP was strongly diminished by the mutation, but k(cat)/K-M,K-PRPP a nd the dissociation constant (K-D,K-PRPP) were nearly unaffected. We conclu de that the proline in the PRPP binding site of UPRTase is of only little i mportance for binding of PRPP to the free enzyme, but is critical for bindi ng of uracil to the enzyme-PRPP complex and for the catalytic rate.