Identification of an Active Site Residue of the R1 Subunit of Ribonucleotide Reductase from Escherichia coli: Characterization of Substrate-Induced Polypeptide Cleavage by C225SR1

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• 作者：Wilfred A. van der Donk ; Chenhui Zeng ; Klaus Biemann ; JoAnne Stubbe ; AnneMarie Hanlon ; and Jack Kyte
• 刊名：Biochemistry
• 出版年：1996
• 出版时间：August 6, 1996
• 年：1996
• 卷：35
• 期：31
• 页码：10058 - 10067
• 全文大小：517K
• 年卷期：v.35,no.31(August 6, 1996)
• ISSN：1520-4995

文摘

Incubation of the C225S mutant of the R1 subunit of ribonucleotidereductase from Escherichiacoli with the R2 subunit and nucleoside diphosphates leads tofragmentation of the polypeptide backboneof R1 [Mao, S. S., Holler, T. P., Bollinger, J. M., Jr., Yu, G. X.,Johnston, M. I., & Stubbe, J. (1992)Biochemistry 31, 9744-9751]. The 26 and60 kDa cleavage fragments were purified to homogeneity.The 26 kDa polypeptide was digested with Lys-C, and the peptideswere partially purified by RP-HPLC.Mass spectrometric analysis (MALDI-TOF) of the HPLC fractionsallowed the identification of theC-terminal peptide. The molecular mass of this peptide (2176)revealed that serine-224 constitutes itsC-terminus, and further analysis of the distribution of itsmonoisotopic masses by FAB-MS indicated thatSer224 possesses a carboxamide rather than a carboxylate group.Treatment of the 60 kDa cleavagefragment with cyanogen bromide and subsequent MALDI-TOF analysis of thepartially RP-HPLC purifiedpeptides yielded a fraction containing its N-terminal peptide.This peptide was digested with trypsin, andthe digestion mixture was purified by HPLC. Analysis of thefractions by MALDI-TOF identified theN-terminal peptide and determined a mass of 2222. This masssuggested valine 226 was the N-terminalresidue (modified by an adduct of 28 mass units). Larger amountsof the C-terminal tetrapeptide of the60 kDa fragment (V₂₂₆LIE₂₂₉) were obtainedby complete digestion of the crude reaction mixture withendoproteinase Glu-C. The peptide mixture was then purified on animmunoadsorbent column containingimmobilized antibodies raised against a synthetic peptide with thesequence KVLIE. After elution of theaffinity-bound peptide, it was analyzed by CID-MS verifying that anadduct of 28 mass units was attachedto valine 226. These results indicated that the amino group ofVal226 is formylated. The localization ofthe residues at the cleavage site of C225SR1 provides a biochemicalidentification of the active site regionof the R1 subunit of RDPR from E. coli. The details ofthe mechanism of cleavage remain to be elucidated.