Wang Susan C, Johnson William H, Czerwinski Robert M, Whitman Christian P
Division of Medicinal Chemistry, College of Pharmacy, The University of Texas, Austin, Texas 78712-1074, USA.
Biochemistry. 2004 Jan 27;43(3):748-58. doi: 10.1021/bi035495u.
4-Oxalocrotonate tautomerase (4-OT) and YwhB, a 4-OT homologue found in Bacillus subtilis, exhibit a low level hydratase activity that converts trans-3-haloacrylates to acetaldehyde, presumably through a malonate semialdehyde intermediate. The mechanism for the initial transformation of the 3-haloacrylate to malonate semialdehyde involves Pro-1 as well as an arginine, two residues that play critical roles in the 4-OT-catalyzed isomerization reaction and the YwhB-catalyzed tautomerization reaction. These residues are also critical for the trans-3-chloroacrylic acid dehalogenase (CaaD)-catalyzed conversion of trans-3-haloacrylates to malonate semialdehyde. Recently, 3-bromo- and 3-chloropropiolate, the acetylene analogues of 3-haloacrylates, were characterized as potent irreversible inhibitors of CaaD due to the covalent modification of the catalytic proline. In view of these observations, an investigation of the behavior of 4-OT and YwhB with the 3-halopropiolates was undertaken. The results show that these compounds are potent irreversible inhibitors of 4-OT and YwhB with Pro-1 being the sole site of covalent modification by 3-bromopropiolate. The inactivation process could involve the enzyme-catalyzed addition of water to the 3-halopropiolate yielding an acyl halide, which would inactivate the enzyme or be initiated by the nucleophilic attack of Pro-1 at the C-3 position of the 3-halopropiolate in a Michael type reaction. The presence of the halogen along with Arg-11 could facilitate both reactions with the latter causing the polarization of the alpha,beta-unsaturated acids. The 3-halopropiolates are the first identified inhibitors of YwhB and confirm the importance of Pro-1 in its mechanism. In addition, the results set the stage for the use of these compounds as mechanistic probes of the primary as well as low level activities of 4-OT and YwhB.
4-草酰巴豆酸互变异构酶（4-OT）以及在枯草芽孢杆菌中发现的4-OT同源物YwhB，表现出较低水平的水化酶活性，该活性可将反式-3-卤代丙烯酸酯转化为乙醛，推测是通过丙二酸半醛中间体进行的。3-卤代丙烯酸酯向丙二酸半醛的初始转化机制涉及Pro-1以及一个精氨酸，这两个残基在4-OT催化的异构化反应和YwhB催化的互变异构反应中起关键作用。这些残基对于反式-3-氯丙烯酸脱卤酶（CaaD）催化的反式-3-卤代丙烯酸酯向丙二酸半醛的转化也至关重要。最近，3-卤代丙烯酸酯的乙炔类似物3-溴和3-氯丙炔酸酯，由于催化脯氨酸的共价修饰，被表征为CaaD的有效不可逆抑制剂。鉴于这些观察结果，对4-OT和YwhB与3-卤代丙炔酸酯的行为进行了研究。结果表明，这些化合物是4-OT和YwhB的有效不可逆抑制剂，Pro-1是3-溴丙炔酸酯共价修饰的唯一位点。失活过程可能涉及酶催化的水加到3-卤代丙炔酸酯上生成酰卤，这会使酶失活，或者由Pro-1在3-卤代丙炔酸酯的C-3位进行亲核攻击引发迈克尔型反应。卤素与Arg-11的存在可能促进这两个反应，后者导致α,β-不饱和酸的极化。3-卤代丙炔酸酯是首次鉴定出的YwhB抑制剂，并证实了Pro-1在其机制中的重要性。此外，这些结果为将这些化合物用作4-OT和YwhB主要以及低水平活性的机制探针奠定了基础。
