双羰基还原酶催化的两步羰基还原反应(英文)

Two step process for diketo-reduction catalyzed by diketoreductase

双羰基还原酶(diketoreductase)选择性催化β,δ-二羰基酯底物还原生成相应的双羟基产物,其对映体过量(ee)和非对映体过量(de)均大于99.5%。为阐明该独特酶促反应的催化机制,研究了双羰基还原酶催化的双羰基反应过程及其反应特征。通过反应历程监测发现在双羰基还原及其逆反应过程中均生成两个单羟基中间产物。这两种单羟基中间产物经反相C18色谱法分离纯化,并采用手性分析鉴定了其立体化学特征。两个中间产物可以作为双羰基还原酶的底物被选择性还原,但具有不同的反应速率。反应温度和底物浓度对中间体的形成和消失有较大的影响。此外,初级稳态动力学显示两个中间产物的消除反应速率也不尽相同。结果表明,双羰基还原酶催化的双羰基还原是一个伴有中间产物的生成与消失的两步反应过程,并且反应速率有所不同。

Diketoreductase can catalyze a double reduction of β,δ-diketo ester to corresponding dihydroxy pro-duct with enantiomeric excess（ee） and diastereomeric excess（de） both greater than 99.5%.In order to explore the catalytic mechanism of this unique enzyme,the present study investigated the diketone reduction process and reaction characteristics by diketoreductase.We found that two mono-hydroxy intermediates were produced during the diketone reduction and its reverse reaction.The two intermediates were further separated by C18 column chromatography and structurally confirmed by chiral HPLC with authentic standards.Two mono-hydroxy intermediates could be served as the substrates for the reduction by diketoreductase with different reaction velocities.The formation and disappearance of intermediates were largely affected by temperature and substrate concentration.In addition,steady state kinetics with the two intermediates showed different reaction rates in their disappearance.Collectively,the results indicate that the diketone reduction undergoes a two-step process with the formation of both intermediates,but the disappearance rates for the two intermediates are slightly different.