Inactivation of Lactobacillus leichmannii ribonucleotide reductase by 2',2'-difluoro-2'-deoxycytidine 5'-triphosphate: covalent modification.
Ribonucleotide reductase (RNR) from Lactobacillus leichmannii, a 76 kDa monomer using adenosylcobalamin (AdoCbl) as a cofactor, catalyzes the conversion of nucleoside triphosphates to deoxynucleotides and is rapidly (<30 s) inactivated by 1 equiv of 2',2'-difluoro-2'-deoxycytidine 5'-triphosphate (F(2)CTP). [1'-(3)H]- and [5-(3)H]F(2)CTP were synthesized and used independently to inactivate RNR. Sephadex G-50 chromatography of the inactivation mixture revealed that 0.47 equiv of a sugar was covalently bound to RNR and that 0.71 equiv of cytosine was released. Alternatively, analysis of the inactivated RNR by SDS-PAGE without boiling resulted in 33% of RNR migrating as a 110 kDa protein. Inactivation of RNR with a mixture of [1'-(3)H]F(2)CTP and [1'-(2)H]F(2)CTP followed by reduction with NaBH(4), alkylation with iodoacetamide, trypsin digestion, and HPLC separation of the resulting peptides allowed isolation and identification by MALDI-TOF mass spectrometry (MS) of a (3)H/(2)H-labeled peptide containing C(731) and C(736) from the C-terminus of RNR accounting for 10% of the labeled protein. The MS analysis also revealed that the two cysteines were cross-linked to a furanone species derived from the sugar of F(2)CTP. Incubation of [1'-(3)H]F(2)CTP with C119S-RNR resulted in 0.3 equiv of sugar being covalently bound to the protein, and incubation with NaBH(4) subsequent to inactivation resulted in trapping of 2'-fluoro-2'-deoxycytidine. These studies and the ones in the preceding paper (DOI: 10.1021/bi9021318 ) allow proposal of a mechanism of inactivation of RNR by F(2)CTP involving multiple reaction pathways. The proposed mechanisms share many common features with F(2)CDP inactivation of the class I RNRs.
Lohman
Gregory J S
GJ
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
Stubbe
Joanne
J
D000478:Alkylation; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D003038:Cobamides / Q000737:chemistry / Q000378:metabolism; D003570:Cytidine Triphosphate / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D043525:Cytosine Deaminase / Q000037:antagonists & inhibitors / Q000138:chemical synthesis; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D052199:Lactobacillus leichmannii / Q000201:enzymology*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D012264:Ribonucleotide Reductases / Q000037:antagonists & inhibitors* / Q000302:isolation & purification / Q000378:metabolism*; D053719:Tandem Mass Spectrometry
D001426:Bacterial Proteins; D003038:Cobamides; D004791:Enzyme Inhibitors; D010446:Peptide Fragments; C054403:2',2'-difluorodeoxycytidine 5'-triphosphate; D003570:Cytidine Triphosphate; D012264:Ribonucleotide Reductases; D043525:Cytosine Deaminase; C000913:cobamamide