Functional and structural characterization of ovine ornithine transcarbamoylase.
Ornithine transcarbamoylase from ovine liver has been purified to homogeneity. Like all anabolic OTCs, the ovine enzyme is a trimer, constituted by identical subunits of 34 kDa. Sequence analysis of the 54 N-terminal residues of ovine OTC shows a high degree of homology with the human enzyme. The optimum pH and the Michaelis constants for the catalytic reaction were determined. The ovine enzyme is the most thermostable one among mammals OTCs, its critical temperature being 6 degrees C higher than those measured for the other enzymes. The enzyme has been crystallised and the structure determined at 3.5 A resolution. Crystals belong to the cubic P4(3)32 space group, with a = b = c = 184.7 A and a solvent content of about 80%. There is no evidence of any ligand in the active site cavity, indicating that the crystals contain an unliganded or T state of the enzyme. The unliganded OTCase enzyme adopts a trimeric structure which, in the crystal, presents a three-fold axis coincident with the crystallographic one. The conformation of each monomer in the trimer is quite similar to that of the liganded human protein, with the exception of a few loops, directly interacting with the substrate(s), which are able to induce a rearrangement of the quaternary organisation of the trimer, that accounts for the cooperative behaviour of the enzyme following the binding of the substrates.
De Gregorio
Ambra
A
Department of Organic and Biological Chemistry, University of Messina, Salita Sperone 31, 98166 Messina, Italy.
Battistutta
Roberto
R
Arena
Nicoletta
N
Panzalorto
Manuela
M
Francescato
Pietro
P
Valentini
Giovanna
G
Bruno
Giuseppe
G
Zanotti
Giuseppe
G
D000818:Animals; D018360:Crystallography, X-Ray; D019281:Dimerization; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008024:Ligands; D008099:Liver / Q000201:enzymology; D008958:Models, Molecular; D009954:Ornithine Carbamoyltransferase / Q000737:chemistry*; D012756:Sheep; D013438:Sulfhydryl Compounds / Q000737:chemistry; D013696:Temperature
D008024:Ligands; D013438:Sulfhydryl Compounds; D009954:Ornithine Carbamoyltransferase