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.
Department of Biochemistry, University of Illinois, Urbana 61801.
J M
JM
Nosal
R L
RL
Switzer
M A
MA
Becker
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