Rosenthal G A, Dahlman D L
Graduate Center for Toxicology, University of Kentucky, Lexington 40506.
J Biol Chem. 1990 Jan 15;265(2):868-73.
Ornithine aminotransferase (L-ornithine:2-oxo-acid aminotransferase (EC 2.6.1.13)) has been purified to homogeneity from last instar larvae of the tobacco hornworm, Manduca sexta (Sphingidae). This enzyme is a 144,000-Da tetramer constructed from 36,000-Da protomeric units. It has a high aspartate/asparagine and glutamate/glutamine content and 2 cysteine residues/subunit. All 8 cysteine residues can react with N-ethylmaleimide to inactivate the enzyme. Maintenance of the enzyme in the presence of 2-mercaptoethanol and dithiothreitol maximizes enzymatic activity and improves storage conditions, presumably by protecting these sulfhydryl groups. The apparent Km values for L-ornithine and 2-oxoglutaric acid are 2.3 and 3.2 mM, respectively. The turnover number is 2.0 +/- 0.1 mumol min-1 mumol-1. L-Canaline (L-2-amino-4-(aminooxy)butyric acid) is a potent ornithine aminotransferase inhibitor. Reaction of the enzyme with L-[U-14C]canaline produces an enzyme-bound, covalently linked, radiolabeled canaline-pyridoxal phosphate oxime. The L-[U-14C]canaline-pyridoxal phosphate oxime has been isolated from canaline-treated enzyme. Dialysis of canaline-inactivated ornithine aminotransferase against free pyridoxal phosphate slowly reactivates the enzyme as the oxime is replaced by pyridoxal phosphate. Analysis of L-[U-14C]canaline binding to ornithine aminotransferase reveals the presence of 4 mol of pyridoxal phosphate/mol of enzyme.
鸟氨酸转氨酶（L-鸟氨酸：2-氧代酸转氨酶（EC 2.6.1.13））已从烟草天蛾（天蛾科）末龄幼虫中纯化至同质。该酶是一种由36,000道尔顿的亚基构成的144,000道尔顿的四聚体。它具有高含量的天冬氨酸/天冬酰胺以及谷氨酸/谷氨酰胺，并且每个亚基含有2个半胱氨酸残基。所有8个半胱氨酸残基均可与N-乙基马来酰亚胺反应使酶失活。在2-巯基乙醇和二硫苏糖醇存在的情况下保存该酶可使酶活性最大化并改善储存条件，这可能是通过保护这些巯基实现的。L-鸟氨酸和2-氧代戊二酸的表观Km值分别为2.3和3.2 mM。周转数为2.0±0.1 μmol min⁻¹ μmol⁻¹。L-刀豆氨酸（L-2-氨基-4-（氨基氧基）丁酸）是一种有效的鸟氨酸转氨酶抑制剂。该酶与L-[U-¹⁴C]刀豆氨酸反应会产生一种与酶结合的、共价连接的、放射性标记的刀豆氨酸-磷酸吡哆醛肟。L-[U-¹⁴C]刀豆氨酸-磷酸吡哆醛肟已从经刀豆氨酸处理的酶中分离出来。将经刀豆氨酸失活的鸟氨酸转氨酶用游离的磷酸吡哆醛进行透析，随着肟被磷酸吡哆醛取代，酶会缓慢重新激活。对L-[U-¹⁴C]刀豆氨酸与鸟氨酸转氨酶结合的分析表明，每摩尔酶存在4摩尔磷酸吡哆醛。
