Ménard R, Khouri H E, Plouffe C, Dupras R, Ripoll D, Vernet T, Tessier D C, Lalberté F, Thomas D Y, Storer A C
Biotechnology Research Institute, National Research Council, Montréal, Québec, Canada.
Biochemistry. 1990 Jul 17;29(28):6706-13. doi: 10.1021/bi00480a021.
The controversy concerning the various suggested roles for the side chain of Asp158 in the active site of papain has been clarified by using site-directed mutagenesis. Both wild-type papain and an Asp158 Asn variant were produced in a baculovirus-insect cell expression system, purified to homogeneity from the culture, and characterized kinetically. With CBZ-Phe-Arg-MCA as substrate, the kcat/KM and kcat values obtained for the Asp158Asn papain are 20,000 M-1.s-1 and 34 s-1, respectively, as compared with values of 120,000 M-1.s-1 and 51 s-1 obtained for the wild-type papain. In addition, the pH-(kcat/KM) profile for the Asp158Asn enzyme is shifted relative to that for the wild-type enzyme to lower values by approximately 0.3 pH unit. This shows clearly that Asp158 is not, as previously postulated, an essential catalytic residue. In addition, the pH dependency data are interpreted to indicate that, contrary to earlier suggestions, the negatively charged side chain of Asp158 does not significantly stabilize the active-site thiolate-imidazolium ion pair. However, its presence does influence the pKa's associated with ion-pair formation in a manner compatible with electrostatic considerations.
通过定点诱变，澄清了关于木瓜蛋白酶活性位点中Asp158侧链各种假定作用的争议。野生型木瓜蛋白酶和Asp158 Asn变体均在杆状病毒 - 昆虫细胞表达系统中产生，从培养物中纯化至同质，并进行动力学表征。以CBZ - Phe - Arg - MCA为底物，Asp158Asn木瓜蛋白酶的kcat/KM和kcat值分别为20,000 M-1·s-1和34 s-1，而野生型木瓜蛋白酶的值为120,000 M-1·s-1和51 s-1。此外，Asp158Asn酶的pH - (kcat/KM)曲线相对于野生型酶的曲线向更低值偏移约0.3个pH单位。这清楚地表明，Asp158并非如先前假设的那样是必需的催化残基。此外，对pH依赖性数据的解释表明，与早期观点相反，Asp158带负电荷的侧链不会显著稳定活性位点硫醇盐 - 咪唑鎓离子对。然而，它的存在确实以与静电考虑相符的方式影响与离子对形成相关的pKa值。
