IDENTIFICATION OF AN ACTIVE-SITE RESIDUE OF THE R1 SUBUNIT OF RIBONUCLEOTIDE REDUCTASE FROM ESCHERICHIA-COLI - CHARACTERIZATION OF SUBSTRATE-INDUCED POLYPEPTIDE CLEAVAGE BY C225SR1

Incubation of the C225S mutant of the R1 subunit of ribonucleotide red uctase from Escherichia coli with the R2 subunit and nucleoside diphos phates leads to fragmentation of the polypeptide backbone of 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 and 60 kDa cleavage fragments were purified to homogeneity. The 26 kDa polypeptid e was digested with Lys-C, and the peptides were partially purified by RP-HPLC. Mass spectrometric analysis (MALDI-TOF) of the HPLC fraction s allowed the identification of the C-terminal peptide, The molecular mass of this peptide (2176) revealed that serine-224 constitutes its C -terminus, and further analysis of the distribution of its monoisotopi c masses by FAB-MS indicated that Ser224 possesses a carboxamide rathe r than a carboxylate group. Treatment of the 60 kDa cleavage fragment with cyanogen bromide and subsequent MALDI-TOF analysis of the partial ly RP-HPLC purified peptides yielded a fraction containing its N-termi nal peptide. This peptide was digested with trypsin, and the digestion mixture was purified by HPLC. Analysis of the fractions by MALDI-TOF identified the N-terminal peptide and determined a mass of 2222. This mass suggested valine 226 was the N-terminal residue (modified by an a dduct of 28 mass units). Larger amounts of the C-terminal tetrapeptide of the 60 kDa fragment (V(226)LIE(229)) were obtained by complete dig estion of the crude reaction mixture with endoproteinase Glu-C. The pe ptide mixture was then purified on an immunoadsorbent column containin g immobilized antibodies raised against a synthetic peptide with the s equence KVLIE. After elution of the affinity-bound peptide, it was ana lyzed by CID-MS verifying that an adduct of 28 mass units was attached to valine 226. These results indicated that the amino group of Val226 is formylated. The localization of the residues at the cleavage site of C225SR1 provides a biochemical identification of the active site re gion of the R1 subunit of RDPR from E. coli, The details of the mechan ism of cleavage remain to be elucidated.