Abstract:
:Ribonucleotide reductase (RDPR) from Escherichia coli is composed of two subunits, R1 and R2, both of which are required to catalyze the conversion of nucleotides to deoxynucleotides. This reduction process is accompanied by oxidation of two cysteines within the active site to a disulfide. One of these putative active site cysteines, C225, has been mutated to a serine, and the properties of this mutant (C225SR1) have been investigated in detail. Incubation of C225SR1 and R2 with [3'-3H,U-14C]UDP results in time-dependent inactivation of the enzyme! This inactivation is accompanied by production of 2.4 uracils, 3H2O, and 3H,14C-labeled protein with an absorbance change at 320 nm. There is an isotope effect (kH/k3H) on uracil production of 3.2. In addition, the tyrosyl radical on R2 is reduced. The observation of 3H2O, indicative of 3' carbon-hydrogen bond cleavage and loss of the tyrosyl radical, provides a direct test of our mechanistic hypothesis that cleavage of this bond occurs concomitantly with tyrosyl radical reduction. Incubation of [3'-2H]UDP with C225SR1 and R2 resulted in a V and V/K isotope effect on loss of the radical of 2.0 and 2.0, respectively. These studies provide the first direct evidence for protein radical involvement in catalysis. Reduction of the tyrosyl radical on R2 is accompanied by a stoichiometric cleavage of the R1 polypeptide into two new polypeptides of 26 and 61 kDa. The 26-kDa polypeptide is the N-terminus of R1, and hence cleavage of the polypeptide is occurring in the region of the mutation. The N-terminus of the 61-kDa polypeptide is blocked.(ABSTRACT TRUNCATED AT 250 WORDS)
journal_name
Biochemistryjournal_title
Biochemistryauthors
Mao SS,Holler TP,Bollinger JM Jr,Yu GX,Johnston MI,Stubbe Jdoi
10.1021/bi00155a030subject
Has Abstractpub_date
1992-10-13 00:00:00pages
9744-51issue
40eissn
0006-2960issn
1520-4995journal_volume
31pub_type
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