Brown A, Nemeria N, Yi J, Zhang D, Jordan W B, Machado R S, Guest J R, Jordan F
Department of Chemistry, Rutgers, The State University of New Jersey, Newark, New Jersey 07102, USA.
Biochemistry. 1997 Jul 1;36(26):8071-81. doi: 10.1021/bi970094y.
A new class of compounds, the 2-oxo-3-alkynoic acids with a phenyl substituent at carbon 4 was reported by the authors as potent irreversible and mechanism-based inhibitors of the thiamin diphosphate- (ThDP-) dependent enzyme pyruvate decarboxylase [Chiu, C.-F., & Jordan, F. (1994) J. Org. Chem. 59, 5763-5766]. The method has been successfully extended to the synthesis of the 4-, 5-, and 7-carbon aliphatic members of this family of compounds. These three compounds were then tested on three ThDP-dependent pyruvate decarboxylases: the Escherichia coli pyruvate dehydrogenase multienzyme complex (PDHc) and its E1 (ThDP-dependent) component, pyruvate oxidase (POX, phosphorylating; from Lactobacillus plantarum),and pyruvate decarboxylase (PDC) from Saccharomycescerevisiae. All three enzymes were irreversibly inhibited by the new compounds. The 4-carbon acid is the best substrate-analog inactivator known to date for PDHc, more potent than either fluoropyruvate or bromopyruvate. The following conclusions were drawn from extensive studies with PDHc: (a) The kinetics of inactivation of PDH complexes and of resolved E1 by 2-oxo-3-alkynoic acids is time- and concentration-dependent. (b) The 4-carbon acid has a Ki 2 orders of magnitude stronger than the 5-carbon acid, clearly demonstrating the substrate specificity of PDHc. (c) The rate of inactivation of PDH complexes and of resolved E1 by 2-oxo-3-alkynoic acids is enhanced by the addition of ThDP and MgCl2. (d) Pyruvate completely protects E1 and partially protects PDHc from inactivation by 2-oxo-3-butynoic acid. (e) E1 but not E2-E3 is the target of inactivation by 2-oxo-3-butynoic acid. (f) Inactivation of E1 by 2-oxo-3-butynoic acid is accompanied by modification of 1.3 cysteines/E1 monomer. The order of reactivity with the 4-carbon acid was PDHc > POX > PDC. While the order of reactivity with PDHc and POX was 2-oxo-3-butynoic acid > 2-oxo-3-pentynoic acid > 2-oxo-3-heptynoic acid, the order of reactivity was reversed with PDC.
作者报道了一类新型化合物，即在碳4位带有苯基取代基的2-氧代-3-炔酸，它们是硫胺素二磷酸（ThDP）依赖性酶丙酮酸脱羧酶的强效不可逆且基于机制的抑制剂[邱，C.-F.，& 乔丹，F.（1994年）《有机化学杂志》59，5763 - 5766]。该方法已成功扩展用于合成该类化合物的4碳、5碳和7碳脂肪族成员。然后对这三种化合物在三种ThDP依赖性丙酮酸脱羧酶上进行了测试：大肠杆菌丙酮酸脱氢酶多酶复合物（PDHc）及其E1（ThDP依赖性）组分、丙酮酸氧化酶（POX，磷酸化型；来自植物乳杆菌）以及酿酒酵母的丙酮酸脱羧酶（PDC）。这三种酶均被这些新化合物不可逆地抑制。4碳酸是迄今为止已知的对PDHc最有效的底物类似物失活剂，比氟丙酮酸或溴丙酮酸更有效。通过对PDHc进行广泛研究得出了以下结论：（a）2-氧代-3-炔酸使PDH复合物和分离出的E1失活的动力学是时间和浓度依赖性的。（b）4碳酸的Ki比5碳酸强2个数量级，清楚地证明了PDHc的底物特异性。（c）添加ThDP和MgCl2可增强2-氧代-3-炔酸使PDH复合物和分离出的E1失活的速率。（d）丙酮酸可完全保护E1，并部分保护PDHc免受2-氧代-3-丁炔酸的失活作用。（e）E1而非E2 - E3是2-氧代-3-丁炔酸失活的靶点。（f）2-氧代-3-丁炔酸使E1失活伴随着每个E1单体有1.3个半胱氨酸被修饰。与4碳酸反应活性由高到低的顺序为PDHc > POX > PDC。虽然与PDHc和POX反应活性由高到低的顺序为2-氧代-3-丁炔酸 > 2-氧代-3-戊炔酸 > 2-氧代-3-庚炔酸，但与PDC反应时该顺序相反。
