Characterization of enzymatic properties of human ribonucleotide reductase holoenzyme reconstituted in vitro from hRRM1, hRRM2, and p53R2 subunits.
Ribonucleotide reductase (RR) is a highly regulated enzyme in the deoxyribonucleotide synthesis pathway. RR is responsible for the de novo conversion of ribonucleoside diphosphates to deoxyribonucleoside diphosphates, which are essential for DNA synthesis and repair. Besides two subunits, hRRM1 and hRRM2, p53R2 is a newly identified member of RR family that is induced by ultraviolet light in a p53-dependent manner. To understand the molecular interaction of RR subunits, we employed a eukaryotic expression system to express and purify all three subunits. After in vitro reconstitution, the results of [(3)H]CDP reduction assay showed that both eukaryotic recombinant hRRM2 and p53R2 proteins could interact with hRRM1 to form functional RR holoenzyme. The reconstituted RR activity was time-dependent and the reaction rate reached the plateau phase after 40min incubation. No matter the concentration, RR holoenzyme reconstituted from p53R2 and hRRM1 could only achieve about 40-75% kinetic activity of that from hRRM2 and hRRM1. The synthetic C-terminal heptapeptide competition assays confirmed that hRRM2 and p53R2 share the same binding site on hRRM1, but the binding site on hRRM1 demonstrated higher affinity for hRRM2 than for p53R2. In allosteric regulation assay, the effect of activation or inhibition of hRRM1 with ATP or dATP suggested that these effectors could regulate RR activity independent of different RR small subunits. Taken together, the eukaryotic expression system RR holoenzyme will provide a very useful tool to understand the molecular mechanisms of RR activity and the interactions of its subunits.
Qiu
Weihua
W
Department of Medical Oncology and Therapeutic Research, City of Hope National Medical Center, Duarte, CA 91010, USA.
Zhou
Bingsen
B
Darwish
Dana
D
Shao
Jimin
J
Yen
Yun
Y
D000494:Allosteric Regulation; D000495:Allosteric Site; D001667:Binding, Competitive; D002460:Cell Line; D005978:Glutathione / Q000235:genetics; D006801:Humans; D007624:KB Cells; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012262:Ribonucleoside Diphosphate Reductase / Q000737:chemistry* / Q000378:metabolism; D012264:Ribonucleotide Reductases / Q000737:chemistry* / Q000378:metabolism*; D025521:Tumor Suppressor Proteins / Q000737:chemistry / Q000502:physiology*
D011993:Recombinant Fusion Proteins; D025521:Tumor Suppressor Proteins; D012264:Ribonucleotide Reductases; C093713:ribonucleotide reductase M2; C475286:RRM1 protein, human; D012262:Ribonucleoside Diphosphate Reductase; D005978:Glutathione