title: Gemcitabine 5'-triphosphate is a stoichiometric mechanism-based inhibitor of Lactobacillus leichmannii ribonucleoside triphosphate reductase: evidence for thiyl radical-mediated nucleotide radical formation.
abstract: Ribonucleoside triphosphate reductase (RTPR) from Lactobacillus leichmannii utilizes adenosylcobalamin and catalyzes the conversion of nucleoside triphosphates to deoxynucleoside triphosphates. One equivalent of 2',2'-difluoro-2'-deoxycytidine 5'-triphosphate, F2dCTP, rapidly inactivates RTPR. Analysis of the reaction products reveals that inactivation is accompanied by release of two fluoride ions and 0.84 equiv of 5'-deoxyadenosine and attachment of 1 equiv of corrin covalently to an active-site cysteine residue of RTPR. No cytosine release was detected. Proteolysis of corrin-labeled RTPR with endoproteinase Glu-C and peptide mapping at pH 5.8 revealed that C419 was predominantly modified. The kinetics of the inactivation have been examined by stopped-flow (SF) UV-vis spectroscopy and rapid freeze quench (RFQ) electron paramagnetic resonance (EPR) spectroscopy. Monitoring DeltaA525 nm shows that cob(II)alamin is formed with an apparent kobs of 50 s-1, only 2. 5-fold slower than a similar experiment carried out with cytidine 5'-triphosphate (CTP). The same reaction mixture was thus quenched at times from 22 ms to 30 s and examined by EPR spectroscopy. At early time points the EPR spectrum resembled a thiyl radical exchange coupled to cob(II)alamin. From 22 to 255 ms the total spin concentration remained unchanged at 1.4 spins/RTPR, twice that predicted by the amount of cob(II)alamin determined by SF. However, with time the signal attributed to the thiyl radical-cob(II)alamin disappears and new signal(s) with broad feature(s) at g = 2.33 and a sharp feature at g = 2.00 appeared, suggesting formation of cob(II)alamin and a nucleotide-based radical with only dipolar interactions. These studies have been interpreted to support the proposal that an RTPR-based thiyl radical can give rise to a nucleotide-based radical.
authors.affiliation: Department of Chemistry and Biochemistry, Brigham Young University, Provo, Utah 84602, USA.
authors.forename: D J
authors.initials: DJ
authors.lastname: Silva
authors.forename: J
authors.initials: J
authors.lastname: Stubbe
authors.forename: V
authors.initials: V
authors.lastname: Samano
authors.forename: M J
authors.initials: MJ
authors.lastname: Robins
mesh_terms: D003596:Cytosine / Q000378:metabolism; D003839:Deoxyadenosines / Q000378:metabolism; D003841:Deoxycytidine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D004578:Electron Spin Resonance Spectroscopy; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D004795:Enzyme Stability / Q000187:drug effects; D005459:Fluorides / Q000378:metabolism; D005609:Free Radicals / Q000378:metabolism; D007778:Lactobacillus / Q000201:enzymology*; D010455:Peptides / Q000378:metabolism; D011489:Protein Denaturation; D012264:Ribonucleotide Reductases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D013438:Sulfhydryl Compounds / Q000494:pharmacology*; D014805:Vitamin B 12 / Q000378:metabolism; D000093542:Gemcitabine
chemical_list: D003839:Deoxyadenosines; D004791:Enzyme Inhibitors; D005609:Free Radicals; D010455:Peptides; D013438:Sulfhydryl Compounds; D003841:Deoxycytidine; C033714:5'-deoxyadenosine; D003596:Cytosine; D012264:Ribonucleotide Reductases; D014805:Vitamin B 12; D005459:Fluorides; D000093542:Gemcitabine