Preugschat F, Averett D R, Clarke B E, Porter D J
Division of Biochemistry, Glaxo Wellcome, Research Triangle Park, North Carolina 27709, USA.
J Biol Chem. 1996 Oct 4;271(40):24449-57. doi: 10.1074/jbc.271.40.24449.
The helicase domain of hepatitis C virus NS3 (genotype 1b) was expressed in Escherichia coli and purified to homogeneity. The purified protein catalyzed the hydrolysis of nucleoside triphosphates (NTP) and the unwinding of duplex RNA in the presence of divalent metal ion. The enzyme was not selective for the NTP substrate. For example, UTP and acyclovir triphosphate were hydrolyzed efficiently by the enzyme. The rate of NTP hydrolysis was stimulated up to 27-fold by oligomeric nucleic acids (NA). Furthermore, NA bound to the enzyme with concomitant quenching of the intrinsic protein fluorescence. The dissociation constants of the enzyme for selected NA in the absence of NTP were between 10 and 35 microM at pH 7.0 and 25 degrees C. The enzyme had maximal affinity for NA with 12 or more nucleotides. A detailed steady-state and pre-steady-state kinetic analysis of ATP hydrolysis was made with (dU)18 as the effector. The kcat values for ATP hydrolysis in the presence and absence of (dU)18 were 80 s-1 and 2.7 s-1, respectively. The association (dissociation) rate constants for the enzyme and (dU)18 in the presence and absence of ATP were 5.7 microM-1 s-1 (3.9 s-1) and 290 microM-1 s-1 (2.27 s-1), respectively. The association (dissociation) rate constants for the enzyme and ATP in the presence and absence of (dU)18 were 0.4 microM-1 s-1 (<0.5 s-1) and 0.9 microM-1 s-1 (<10(-1) s-1), respectively. These data were consistent with a random kinetic mechanism.
丙型肝炎病毒NS3（1b基因型）的解旋酶结构域在大肠杆菌中表达并纯化至同质。纯化后的蛋白质在二价金属离子存在的情况下催化核苷三磷酸（NTP）的水解以及双链RNA的解旋。该酶对NTP底物没有选择性。例如，UTP和阿昔洛韦三磷酸可被该酶有效水解。寡聚核酸（NA）可将NTP水解速率提高至27倍。此外，NA与该酶结合，同时使蛋白质固有荧光淬灭。在pH 7.0和25℃条件下，该酶对选定NA在无NTP时的解离常数在10至35微摩尔之间。该酶对含12个或更多核苷酸的NA具有最大亲和力。以（dU）18作为效应物，对ATP水解进行了详细的稳态和预稳态动力学分析。在有和无（dU）18存在的情况下，ATP水解的kcat值分别为80 s-1和2.7 s-1。在有和无ATP存在的情况下，该酶与（dU）18的缔合（解离）速率常数分别为5.7微摩尔-1 s-1（3.9 s-1）和290微摩尔-1 s-1（2.27 s-1）。在有和无（dU）18存在的情况下，该酶与ATP的缔合（解离）速率常数分别为0.4微摩尔-1 s-1（<0.5 s-1）和0.9微摩尔-1 s-1（<10-1 s-1）。这些数据与随机动力学机制一致。
