Kaartinen V, Williams J C, Tomich J, Yates J R, Hood L E, Mononen I
Division of Medical Genetics, Children's Hospital of Los Angeles, California 90054-0700.
J Biol Chem. 1991 Mar 25;266(9):5860-9.
The apparent active site of human leukocyte glycoasparaginase (N4-(beta-acetylglucosaminyl)-L-asparaginase EC 3.5.1.26) has been studied by labeling with an asparagine analogue, 5-diazo-4-oxo-L-norvaline. Glycoasparaginase was purified 4,600-fold from human leukocytes with an overall recovery of 12%. The purified enzyme has a Km of 110 microM, a Vmax of 34 mumol x l-1 x min-1, and a specific activity of 2.2 units/mg protein with N4-(beta-N-acetylglucosaminyl)-L-asparagine as substrate. The carbohydrate content of the enzyme is 15%, and it exhibits a broad pH maximum between 7 and 9. The 88-kDa native enzyme is composed of 19-kDa light (L) chains and 25-kDa heavy (H) chains and it has a heterotetrameric structure of L2H2-type. The glycoasparaginase activity decreases rapidly and irreversibly in the presence of 5-diazo-4-oxo-L-norvaline. At any one concentration of the compound, the inactivation of the enzyme is pseudo-first-order with time. The inhibitory constant, K1, is 80 microM and the second-order rate constant 1.25 x 10(3) M-1 min-1 at pH 7.5. The enzyme activity is competitively protected against this inactivation by its natural substrate, aspartylglucosamine, indicating that this inhibitor binds to the active site or very close to it. The covalent incorporation of [5-14C]diazo-4-oxo-L-norvaline paralleled the loss of the enzymatic activity and one inhibitor binding site was localized to each L-subunit of the heterotetrameric enzyme. Four peptides with the radioactive label were generated, purified by high performance liquid chromatography, and sequenced by Edman degradation. The sequences were overlapping and all contained the amino-terminal tripeptide of the L-chain. By mass spectrometry, the reacting group of 5-diazo-4-oxo-L-norvaline was characterized as 4-oxo-L-norvaline that was bound through an alpha-ketone ether linkage to the hydroxyl group of the amino-terminal amino acid threonine.
通过用天冬酰胺类似物5-重氮-4-氧代-L-正缬氨酸进行标记，对人白细胞糖天冬酰胺酶（N4-(β-乙酰氨基葡萄糖基)-L-天冬酰胺酶，EC 3.5.1.26）的表观活性位点进行了研究。从人白细胞中纯化糖天冬酰胺酶，纯化倍数达4600倍，总回收率为12%。纯化后的酶以N4-(β-N-乙酰氨基葡萄糖基)-L-天冬酰胺为底物时，Km为110μM，Vmax为34μmol·L⁻¹·min⁻¹，比活性为2.2单位/mg蛋白。该酶的碳水化合物含量为15%，在pH 7至9之间呈现较宽的最适pH范围。88 kDa的天然酶由19 kDa的轻（L）链和25 kDa的重（H）链组成，具有L2H2型异源四聚体结构。在5-重氮-4-氧代-L-正缬氨酸存在下，糖天冬酰胺酶活性迅速且不可逆地降低。在该化合物的任何一个浓度下，酶的失活呈假一级反应。在pH 7.5时，抑制常数K1为80μM，二级速率常数为1.25×10³ M⁻¹·min⁻¹。其天然底物天冬氨酰葡萄糖可竞争性地保护酶活性免受这种失活影响，这表明该抑制剂结合在活性位点或其非常接近的位置。[5-¹⁴C]重氮-4-氧代-L-正缬氨酸的共价掺入与酶活性的丧失平行，且一个抑制剂结合位点定位于异源四聚体酶的每个L亚基上。产生了四个带有放射性标记的肽段，通过高效液相色谱法进行纯化，并通过埃德曼降解法进行测序。这些序列相互重叠，且均包含L链的氨基末端三肽。通过质谱分析，5-重氮-4-氧代-L-正缬氨酸的反应基团被鉴定为4-氧代-L-正缬氨酸，它通过α-酮醚键与氨基末端氨基酸苏氨酸的羟基相连。
