Radiation inactivation of ribonucleotide reductase, an enzyme with a stable free radical.
Herpes simplex virus ribonucleotide reductase (RR) is a tetrameric enzyme composed of two homodimers of large R1 and small R2 subunits with a tyrosyl free radical located on the small subunit. Irradiation of the holoenzyme yielded simple exponential decay curves and an estimated functional target size of 315 kDa. Western blot analysis of irradiated holoenzyme R1 and R2 yielded target sizes of 281 kDa and 57 kDa (approximately twice their expected size). Irradiation of free R1 and analysis by all methods yielded a single exponential decay with target sizes ranging from 128-153 kDa. For free R2, quantitation by enzyme activity and Western blot analyses yielded simple inactivation curves but considerably different target sizes of 223 kDa and 19 kDa, respectively; competition for radioligand binding in irradiated R2 subunits yielded two species, one with a target size of approximately 210 kDa and the other of approximately 20 kDa. These results are consistent with a model in which there is radiation energy transfer between the two monomers of both R1 and R2 only in the holoenzyme, a radiation-induced loss of free radical only in the isolated R2, and an alteration of the tertiary structure of R2.
Bolger
G
G
Department of Biological Sciences, Boehringer Ingelheim (Canada) Limited, Bio-Méga Research Division, Laval, Québec H7S 2G5, Canada.
Liuzzi
M
M
Krogsrud
R
R
Scouten
E
E
McCollum
R
R
Welchner
E
E
Kempner
E
E
D055592:Biophysical Phenomena; D001703:Biophysics; D004735:Energy Transfer; D005609:Free Radicals / Q000737:chemistry; D018259:Herpesvirus 1, Human / Q000201:enzymology; D020035:Holoenzymes / Q000737:chemistry / Q000528:radiation effects; D008970:Molecular Weight; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000528:radiation effects; D012264:Ribonucleotide Reductases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000528:radiation effects*
D005609:Free Radicals; D020035:Holoenzymes; D021122:Protein Subunits; D011994:Recombinant Proteins; D012264:Ribonucleotide Reductases