Johnson W H, Czerwinski R M, Fitzgerald M C, Whitman C P
Medicinal Chemistry Division, College of Pharmacy, The University of Texas, Austin, Texas 78712, USA.
Biochemistry. 1997 Dec 16;36(50):15724-32. doi: 10.1021/bi971608w.
The compound, 2-oxo-3-pentynoate, has been synthesized and tested as an inhibitor of the enzyme 4-oxalocrotonate tautomerase. The enzyme is rapidly and irreversibly inactivated by the acetylenic product analogue in a time-dependent fashion. The enzyme displays saturation kinetics and is protected from inactivation by the presence of substrate. These observations are consistent with inactivation taking place at the active site. Partial reactivation ( approximately 18%) occurs by incubating the inactivated enzyme with 10 mM hydroxylamine (pH 7.3). The partition ratio, determined to be approximately 0.4, suggests that the inactivation of 4-OT by 2-oxo-3-pentynoate shows half-of-the-sites stoichiometry. The same phenomenon is observed in the inactivation of 4-OT by 3-bromopyruvate and can be explained by examination of the crystal structure. Mass spectral analysis shows that a single residue is modified on the enzyme which has been localized to the nine residue amino-terminal fragment Pro-1 to Glu-9. It can be reasonably concluded that Pro-1 is the site of covalent attachment. Inactivation of 4-OT can occur by either a Michael addition of 4-OT to C-4 of 2-oxo-3-pentynoate or by the enzyme-catalyzed rearrangement of 2-oxo-3-pentynoate to an allene derivative which alkylates Pro-1. These results provide the foundation for the use of 2-oxo-3-pentynoate in future mechanistic studies and as a ligand in an inactivated 4-OT complex that can be studied by X-ray crystallography. Finally, 2-oxo-3-pentynoate is an acetylene analogue of a variety of 2-oxo acids and as such may have general utility as an inhibitor of reactions that bind and process these compounds.
化合物2-氧代-3-戊炔酸酯已被合成并作为4-氧代巴豆酸互变异构酶的抑制剂进行了测试。该炔类产物类似物能以时间依赖性方式使该酶迅速且不可逆地失活。该酶呈现饱和动力学，并且底物的存在可保护其不被失活。这些观察结果与失活发生在活性位点一致。通过将失活的酶与10 mM羟胺（pH 7.3）孵育，可发生部分重新激活（约18%）。分配比测定为约0.4，表明2-氧代-3-戊炔酸酯使4-OT失活呈现半位点化学计量关系。在3-溴丙酮酸使4-OT失活的过程中也观察到了相同现象，并且通过对晶体结构的研究可以对此进行解释。质谱分析表明，酶上有一个单一残基被修饰，该残基已定位到九残基的氨基末端片段Pro-1至Glu-9。可以合理推断Pro-1是共价连接的位点。4-OT的失活可能是通过4-OT对2-氧代-3-戊炔酸酯的C-4进行迈克尔加成，或者是通过酶催化2-氧代-3-戊炔酸酯重排为丙二烯衍生物，该衍生物使Pro-1烷基化。这些结果为未来在机理研究中使用2-氧代-3-戊炔酸酯以及作为可通过X射线晶体学研究的失活4-OT复合物中的配体奠定了基础。最后，2-氧代-3-戊炔酸酯是多种2-氧代酸的乙炔类似物，因此可能作为结合和处理这些化合物的反应的抑制剂具有普遍用途。
