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Biochemical Characterization of a Human Ribonucleotide Reductase ( Hs RNR) Variant Lacking Activity Regulation
Author(s) -
Rios Paulina,
Perez Goncalves Gerardo,
Miller Kelsey R.,
Drennan Catherine
Publication year - 2022
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2022.36.s1.0r354
Subject(s) - ribonucleotide reductase , protein subunit , ribonucleotide , allosteric regulation , mutagenesis , biochemistry , cofactor , chemistry , active site , deoxyribonucleotides , enzyme , stereochemistry , biology , nucleotide , mutation , gene
Ribonucleotide reductase (RNR) is responsible for the synthesis of deoxyribonucleotides used in DNA synthesis and repair. Class Ia RNRs are composed of two dimeric subunits: a catalytic subunit (α 2 ) which contains the active site and two allosteric regulation sites, and radical generating (β 2 ) subunit housing the diferric‐tyrosyl radical cofactor. The two subunits form an α 2 β 2 heterotetrametric complex that allows the radical to travel from β to α subunit for catalysis. RNR activity is regulated by the binding of ATP/dATP to an N‐terminal cone domain. Human RNRs ( Hs RNRs) are class Ia enzymes that form a homohexameric α 6 in the presence of either dATP or ATP. Although, both α 6 assemblies are morphologically identical, only the α 6 ‐ATP can be disturbed by the addition of β to form the α 2 β 2 complex necessary for catalysis. Recent studies have shown that α subunits contact each other via their cone domains in the α 6 rings, but it is unknown if the cone domains contact β 2 in the α 2 β 2 active complex. Here, we aim to probe the role of the cone domains in catalysis through biochemical and biophysical studies of an α subunit variant lacking the cone domain. We used site‐directed mutagenesis to delete residues 2‐92 of α, and expressed and purified the variant a protein. The variant protein was used for HPLC‐MS based activity assays of Hs RNR. Preliminary results show the cone domain is important for catalysis. Understanding more about RNR’s structure and activity regulation can facilitate development of new cancer therapies.