Clark D D, Allen J R, Ensign S A
Department of Chemistry and Biochemistry, Utah State University, Logan, Utah 84322-0300, USA.
Biochemistry. 2000 Feb 15;39(6):1294-304. doi: 10.1021/bi992282p.
The bacterial metabolism of propylene proceeds by epoxidation to epoxypropane followed by carboxylation to acetoacetate. Epoxypropane carboxylation is a minimetabolic pathway that requires four enzymes, NADPH, NAD(+), and coenzyme M (CoM; 2-mercaptoethanesulfonate) and occurs with the overall reaction stoichiometry: epoxypropane + CO(2) + NADPH + NAD(+) + CoM --> acetoacetate + H(+) + NADP(+) + NADH + CoM. The terminal enzyme of the pathway is NADPH:2-ketopropyl-CoM [2-(2-ketopropylthio)ethanesulfonate] oxidoreductase/carboxylase (2-KPCC), an FAD-containing enzyme that is a member of the NADPH:disulfide oxidoreductase family of enzymes and that catalyzes the reductive cleavage and carboxylation of 2-ketopropyl-CoM to form acetoacetate and CoM according to the reaction: 2-ketopropyl-CoM + NADPH + CO(2) --> acetoacetate + NADP(+) + CoM. In the present work, 2-KPCC has been characterized with respect to the above reaction and four newly discovered partial reactions of relevance to the catalytic mechanism, and each of which requires the formation of a stabilized enolacetone intermediate. These four reactions are (1) NADPH-dependent cleavage and protonation of 2-ketopropyl-CoM to form NADP(+), CoM, and acetone, a reaction analogous to the physiological reaction but in which H(+) is the electrophile; (2) NADP(+)-dependent synthesis of 2-ketopropyl-CoM from CoM and acetoacetate, the reverse of the physiologically important forward reaction; (3) acetoacetate decarboxylation to form acetone and CO(2); and (4) acetoacetate/(14)CO(2) exchange to form (14)C(1)-acetoacetate and CO(2). Acetoacetate decarboxylation and (14)CO(2) exchange occurred independent of NADP(H) and CoM, demonstrating that these substrates are not central to the mechanism of enolate generation and stabilization. 2-KPCC did not uncouple NADPH oxidation or NADP(+) reduction from the reactions involving cleavage or formation of 2-ketopropyl-CoM. N-Ethylmaleimide inactivated the reactions forming/using 2-ketopropyl-CoM but did not inactivate acetoacetate decarboxylation or (14)CO(2) exchange reactions. The biochemical characterization of 2-KPCC and the associated five catalytic activities has allowed the formulation of an unprecedented mechanism of substrate activation and carboxylation that involves NADPH oxidation, a redox active disulfide, thiol-mediated reductive cleavage of a C-S thioether bond, the formation of a CoM:cysteine mixed disulfide, and enolacetone stabilization.
丙烯的细菌代谢过程是先通过环氧化生成环氧丙烷，然后羧化生成乙酰乙酸。环氧丙烷羧化是一条最小代谢途径，需要四种酶、NADPH、NAD(+)和辅酶M（CoM；2-巯基乙烷磺酸盐），其总反应化学计量式为：环氧丙烷 + CO₂ + NADPH + NAD(+) + CoM → 乙酰乙酸 + H⁺ + NADP(+) + NADH + CoM。该途径的末端酶是NADPH:2-酮丙基-CoM [2-(2-酮丙基硫基)乙烷磺酸盐]氧化还原酶/羧化酶（2-KPCC），这是一种含FAD的酶，属于NADPH:二硫化物氧化还原酶家族，它催化2-酮丙基-CoM的还原裂解和羧化反应，生成乙酰乙酸和CoM，反应式为：2-酮丙基-CoM + NADPH + CO₂ → 乙酰乙酸 + NADP(+) + CoM。在本研究中，对2-KPCC在上述反应以及与催化机制相关的四个新发现的部分反应方面进行了表征，每个反应都需要形成稳定的烯醇丙酮中间体。这四个反应分别是：(1) 2-酮丙基-CoM的NADPH依赖性裂解和质子化，生成NADP(+)、CoM和丙酮，该反应类似于生理反应，但其中H⁺是亲电试剂；(2) 由CoM和乙酰乙酸进行的NADP(+)依赖性2-酮丙基-CoM合成，这是生理上重要的正向反应的逆反应；(3) 乙酰乙酸脱羧生成丙酮和CO₂；(4) 乙酰乙酸/(¹⁴)CO₂交换生成(¹⁴)C₁-乙酰乙酸和CO₂。乙酰乙酸脱羧和(¹⁴)CO₂交换反应的发生不依赖于NADP(H)和CoM，这表明这些底物对于烯醇盐的生成和稳定机制并非核心要素。2-KPCC在涉及2-酮丙基-CoM裂解或形成的反应中，并未使NADPH氧化或NADP(+)还原解偶联。N-乙基马来酰亚胺使形成/使用2-酮丙基-CoM的反应失活，但未使乙酰乙酸脱羧或(¹⁴)CO₂交换反应失活。2-KPCC及其相关的五种催化活性的生化表征，使得能够构建一种前所未有的底物活化和羧化机制理论，该机制涉及NADPH氧化、一种氧化还原活性二硫化物、硫醇介导的C-S硫醚键还原裂解、CoM:半胱氨酸混合二硫化物的形成以及烯醇丙酮的稳定化。
