Subunit-subunit interactions in trimeric arginase. Generation of active monomers by mutation of a single amino acid.
The structure of the trimeric, manganese metalloenzyme, rat liver arginase, has been previously determined at 2.1-A resolution (Kanyo, Z. F., Scolnick, L. R., Ash, D. E., and Christianson, D. W., (1996) Nature 383, 554-557). A key feature of this structure is a novel S-shaped oligomerization motif at the carboxyl terminus of the protein that mediates approximately 54% of the intermonomer contacts. Arg-308, located within this oligomerization motif, nucleates a series of intramonomer and intermonomer salt links. In contrast to the trimeric wild-type enzyme, the R308A, R308E, and R308K variants of arginase exist as monomeric species, as determined by gel filtration and analytical ultracentrifugation, indicating that mutation of Arg-308 shifts the equilibrium for trimer dissociation by at least a factor of 10(5). These monomeric arginase variants are catalytically active, with k(cat)/K(m) values that are 13-17% of the value for wild-type enzyme. The arginase variants are characterized by decreased temperature stability relative to the wild-type enzyme. Differential scanning calorimetry shows that the midpoint temperature for unfolding of the Arg-308 variants is in the range of 63.6-65.5 degrees C, while the corresponding value for the wild-type enzyme is 70 degrees C. The three-dimensional structure of the R308K variant has been determined at 3-A resolution. At the high protein concentrations utilized in the crystallizations, this variant exists as a trimer, but weakened salt link interactions are observed for Lys-308.
Department of Biochemistry, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA.
L T
LT
Lavulo
T M
TM
Sossong
M R
MR
Brigham-Burke
M L
ML
Doyle
J D
JD
Cox
D W
DW
Christianson
D E
DE
Ash
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D001119:Arginase / Q000737:chemistry* / Q000235:genetics*; D001120:Arginine / Q000737:chemistry; D002152:Calorimetry, Differential Scanning; D002384:Catalysis; D002850:Chromatography, Gel; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D019281:Dimerization; D004578:Electron Spin Resonance Spectroscopy; D004583:Electrons; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008345:Manganese / Q000737:chemistry; D051379:Mice; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D051381:Rats; D013696:Temperature; D014461:Ultracentrifugation; D014981:Xenopus
D000596:Amino Acids; D008345:Manganese; D001120:Arginine; D001119:Arginase