IDENTIFICATION OF AMINO-ACID-RESIDUES LINKED TO DIFFERENT PROPERTIES OF PHOSPHORIBOSYLPYROPHOSPHATE SYNTHETASE ISOFORM-I AND ISOFORM-II

The catalytic subunit of rat liver phosphoribosylpyrophosphate synthet ase is composed of two isoforms, PRS I and PRS II. The amino-acid sequ ences differ only by 13 residues, out of which two Lys residues of PRS I at positions 4 and 152 give net additional positive charges to PRS I. Previous work has shown that PRS I is more sensitive to inhibition by ADP and GDP and more stable to heat treatment than is PRS II. To id entify amino-acid residues responsible for the different properties, f ive chimeric enzymes between rat PRS I and PRS II and two mutated enzy mes with a single point mutation at position 152 were constructed; the se enzymes were produced in Escherichia coli. Changing Lys-4 of PRS I to Val, together with Ile-5 to Leu, completely abolished sensitivity t o GDP inhibition of PRS I, indicating that Lys-4 in PRS I is critical for GDP inhibition. The substitutions at position 152 had little effec t on GDP inhibition. Characterization of the chimeric enzymes revealed that residues between residues 54-110 and 229-317, namely, Val-55 and /or Ala-81, and Arg-242 and/or Cys-264 of PRS I also contribute to the strong GDP inhibition. Lys-4 was also important for the strong ADP in hibition of PRS I. Regarding the physical properties, chimeric enzymes bearing residues 12-53 of PRS I were stable at 49 degrees C and with digestion with papain and proteinase K. Our observations suggest that Lys-17, Ile-18, and/or Cys-40 of PRS I contribute to stability of the enzyme.