Rhodococcus sp．R04BphD催化C－C断裂的动力学分析

Kinetic Analysis of C-C Cleavage Catalyzed by the Rhodococcus sp. R04 BphD

2-羟基-6-氧-6-苯基己-2,4-二烯酸水解酶(BphD)是一种多氯联苯微生物降解途径中的关键酶.本文通过紫外-可见光光谱分别对突变酶S110A和H265A催化过程中酶-底物复合物进行检测,同时利用停流光谱技术对BphD及其突变酶(S110A、H265A和W266A)催化底物2-羟基-6-氧-6-苯基己-2,4-二烯酸(HOPDA)前稳态动力学进行了研究.结果表明,在BphD催化C-C断裂过程中,产物2-羟基戊-2,4-二烯酸(HPD)迅速生成,其速率常数为22 S-1.底物的消耗(速率常数,220.22 S-1和80.3 S-1)及酶-底物复合物的变化(速率常数,555.56 S-1和66.4 S-1)表明该酶催化过程包括2个动力学阶段:快速底物酮基化作用和C-C键断裂过程.紫外-可见光光谱扫描结果显示,在突变酶S110A的催化过程中,酶-底物复合物在492 nm及510 nm处有最大光吸收,而在突变酶H265A催化中,却没有相似的光吸收,只是在480 nm产生1个新肩峰.BphD及其突变酶S110A、H265A和W266A动力学分析表明,Ser-110主要负责底物C-C键断裂;His-265负责底物由烯醇式向酮式转变,并且与Ser-110和Trp-266共同参与了随后的C-C键断裂过程.结果揭示,除了传统的催化三联体(Ser-110,Asp-237,His-265)外,Trp-266在该水解酶催化反应中也发挥非常重要的作用,这一发现丰富了C-C水解酶的反应动力学机制.

BphD （2-hydroxyl-6-oxo-6-phenylhexa-2,4-dienoie acid hydrolase） is a key enzyme in the pathway of microbial degradation of polychlorinated biphenyls （PCBs）. In the present study, we measured the enzyme-substrate complex （E： S） of mutant enzymes S110A and H265A for their pre-steady state kinetics by stop/flow UV/visible spectrophotometry. The results were simulated by curve-fitting to appropriate models. The pre-steady-state analysis showed that the product formed rapidly with a rate constant of 22 S-1 and the substrate consumption rate were 220.22 S-1 and 80.3 S-1 for the mutants. The formation and dissociation of the enzyme-substrate complex were detected with the rate constants of 555.56 S-1 and 66.4 S-1. The data suggested that the reaction catalyzed by BphD included two kinetic steps： the fast ketonisation and the C-C cleavage. The UV/visible spectrum showed that the Sll0A mutant bound to HOPDA （2-hydroxyl-6-oxo-6-phenylhexa-2,4-dienoic acid） and formed an enzyme substrate complex with a maximum absorption at 492 nm and 510 nm. In the case of H265A, only asmall absorption peak was observed at 480 nm. We concluded that the Ser-ll0 of BphD played a key role in the course of C-C cleavage, while the His-265 was responsible for ketonisation of the substrate, and might also participated in C-C cleavage.