Overexpression, purification, and characterization of recombinant human 5-phosphoribosyl-1-pyrophosphate synthetase isozymes I and II.
Human 5-phosphoribosyl-1-pyrophosphate synthetase isozymes I and II (PRSI and PRSII) have been isolated independently and characterized in pure form. cDNAs for PRSI and PRSII were overexpressed in an Escherichia coli strain which lacks the bacterial 5-phosphoribosyl-1-pyrophosphate synthetase. The recombinant isoforms were purified to virtual homogeneity with specific activities of 25.0 and 35.7 units/mg, respectively, values which are 5-10-fold higher than any previously reported for this enzyme from human sources. Despite 95% amino acid sequence identity, the isoforms differed significantly in several physical and kinetic properties. PRSII was more sensitive to heat inactivation at 55 degrees C and more susceptible to disaggregation to inactive forms in the absence of Mg2+ and ATP than was PRSI. The isoforms were separable on the basis of isoelectric point. PRSI and PRSII also differed significantly in Km values for MgATP and ribose 5-phosphate, pH optima, and Mg2+ and Pi activation curves. PRSII was less sensitive to feedback inhibition by purine nucleotides and more sensitive to inhibition by 2,3-diphosphoglycerate than PRSI. Differences in kinetic properties between PRSI and PRSII are consistent with the suggestion that PRSII predominates in rapidly proliferating cells.
Nosal
J M
JM
Department of Biochemistry, University of Illinois, Urbana 61801.
Switzer
R L
RL
Becker
M A
MA
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D004247:DNA / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development; D006153:Guanosine Diphosphate / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012268:Ribose-Phosphate Pyrophosphokinase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013816:Thermodynamics
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D006153:Guanosine Diphosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D004247:DNA; D012268:Ribose-Phosphate Pyrophosphokinase