氟二硝基苯对乳酸氧化酶的失活与修饰
Inactivation and modification of lactate oxidase with fluorodinitrobenzene.
作者信息
Soon C Y, Shepherd M G, Sullivan P A
出版信息
Biochem J. 1978 Jul 1;173(1):255-62. doi: 10.1042/bj1730255.
Abstract
- Dinitrophenylation of 2 +/- 0.2mol of residues/mol of enzyme-bound FMN resulted in the complete inactivation of the flavoenzyme L-lactate oxidase. 2. Hydrolysates of the inactivated enzyme contained 1mol each of Nim-Dnp-histidine (abbreviation: Dnp-,2,4-dinitrophenyl-; Nim indicates that either of the N atoms in the imidazole ring is substituted) and epsilon-Dnp-lysine/mol of FMN. 3. Competitive inhibitors decreased the extent of inactivation to a 10% loss of activity, and dinitrophenylation was decreased from 2 to approx. 0.5mol/mol of FMN. Only Nim-Dnp-histidine was detected in the hydrolysates. 4. Although the dinitrophenylated enzyme did not possess enzyme activitiy, L-lactate reduced approx. 50% of the enzyme-bound flavin slowly (0.6min-1), and approx. 50% of the flavin in the modified enzyme-bound flavin slowly (0.6min-1), and approx. 50% of the flavin in the modified enzyme formed a complex with bisulphite. 6. The modified enzyme (2mol of Dnp/mol of FMN) was unable to bind substrate analogues and competitive inhibitors.
摘要
- 每摩尔与酶结合的黄素单核苷酸（FMN）中有2±0.2摩尔残基发生二硝基苯基化，导致黄素酶L-乳酸氧化酶完全失活。2. 失活酶的水解产物中，每摩尔FMN含有1摩尔Nim-二硝基苯基组氨酸（缩写：Dnp-，2,4-二硝基苯基-；Nim表示咪唑环中的任意一个氮原子被取代）和ε-二硝基苯基赖氨酸。3. 竞争性抑制剂使失活程度降低至活性损失10%，二硝基苯基化从每摩尔FMN 2摩尔降至约0.5摩尔。水解产物中仅检测到Nim-二硝基苯基组氨酸。4. 尽管二硝基苯基化的酶不具有酶活性，但L-乳酸能缓慢还原约50%与酶结合的黄素（0.6分钟-1），且约50%修饰酶结合的黄素能缓慢还原（0.6分钟-1），并且修饰酶中约50%的黄素与亚硫酸氢盐形成复合物。6. 修饰酶（每摩尔FMN 2摩尔Dnp）无法结合底物类似物和竞争性抑制剂。
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