Inactivation of Lactobacillus leichmannii Ribonucleotide Reductase by 2′,2′-Difluoro-2′-deoxycytidine 5′-Triphosphate: Covalent Modification

被引:9
|
作者
Lohman, Gregory J. S. [1 ]
Stubbe, JoAnne [1 ,2 ]
机构
[1] MIT, Dept Chem, Cambridge, MA 02139 USA
[2] MIT, Dept Biol, Cambridge, MA 02139 USA
基金
美国国家卫生研究院;
关键词
CELL LUNG-CANCER; COBALT BOND HOMOLYSIS; TRIPHOSPHATE REDUCTASE; ESCHERICHIA-COLI; CYSTEINE RESIDUES; PANCREATIC-CANCER; RADICAL FORMATION; SOLID TUMORS; GEMCITABINE; MECHANISM;
D O I
10.1021/bi902132u
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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) by I equiv of 2',2'-difluoro-2'-deoxycytidine 5'-triphosphate (F2CTP). [1'-H-3]- and [5-H-3]F2CTP 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'-H-3]F2CTP and [1'-H-2]F2CTP followed by reduction with NaBH4, alkyl, iodoacetamide, trypsin digestion, and HPLC separation of the resulting peptides allowed isolation and identification by MALDI-TOF mass spectrometry (MS) of a H-3/H-2-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 F2CTP. Incubation of [1'-H-3]F2CTP with C119S-RNR resulted in 0.3 equiv Of Sugar being covalently bound to the protein, and incubation with NaBH4 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 F2CTP involving multiple reaction pathways. The proposed mechanisms share many common features with F2CDP inactivation of the class I RNRs.
引用
收藏
页码:1404 / 1417
页数:14
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