Identification of amino-acid residues linked to different properties of phosphoribosylpyrophosphate synthetase isoforms I and II.
The catalytic subunit of rat liver phosphoribosylpyrophosphate synthetase is composed of two isoforms, PRS I and PRS II. The amino-acid sequences 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 identify amino-acid residues responsible for the different properties, five chimeric enzymes between rat PRS I and PRS II and two mutated enzymes with a single point mutation at position 152 were constructed; these enzymes were produced in Escherichia coli. Changing Lys-4 of PRS I to Val, together with Ile-5 to Leu, completely abolished sensitivity to GDP inhibition of PRS I, indicating that Lys-4 in PRS I is critical for GDP inhibition. The substitutions at position 152 had little effect 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 inhibition 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.
Ahmad
I
I
Department of Biochemistry, Chiba University School of Medicine, Japan.
Ishijima
S
S
Kita
K
K
Tatibana
M
M
D000596:Amino Acids / Q000032:analysis*; D001483:Base Sequence; D002678:Chimera; D003001:Cloning, Molecular; D010450:Endopeptidases; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D007527:Isoenzymes / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000494:pharmacology; D017354:Point Mutation; D012268:Ribose-Phosphate Pyrophosphokinase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification
D000596:Amino Acids; D007527:Isoenzymes; D009711:Nucleotides; D012268:Ribose-Phosphate Pyrophosphokinase; D010450:Endopeptidases