Ordoukhanian Phillip, Joyce Gerald F
Departments of Chemistry, The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
J Am Chem Soc. 2002 Oct 23;124(42):12499-506. doi: 10.1021/ja027467p.
In vitro evolution methods were used to obtain DNA enzymes that cleave either a 2',5'-phosphodiester following a D-ribonucleotide or a 3',5'-phosphodiester following an L-ribonucleotide. Both enzymes can operate in an intermolecular reaction format with multiple turnover. The DNA enzyme that cleaves a 2',5'-phosphodiester exhibits a k(cat) of approximately 0.01 min(-1) and catalytic efficiency, k(cat)/K(m), of approximately 10(8) M(-1) min(-1). The enzyme that cleaves an L-ribonucleotide is about 10-fold slower and has a catalytic efficiency of approximately 4 x 10(5) M(-1) min(-1). Both enzymes require a divalent metal cation for their activity and have optimal catalytic rate at pH 7-8 and 35-50 degrees C. In a comparison of each enzyme's activity with either its corresponding substrate that contains an unnatural ribonucleotide or a substrate that instead contains a standard ribonucleotide, the 2',5'-phosphodiester-cleaving DNA enzyme exhibited a regioselectivity of 6000-fold, while the L-ribonucleotide-cleaving DNA enzyme exhibited an enantioselectivity of 40-fold. These molecules demonstrate how in vitro evolution can be used to obtain regio- and enantioselective catalysts that exhibit specificities for nonnatural analogues of biological compounds.
体外进化方法被用于获得能够切割D - 核糖核苷酸后的2',5'-磷酸二酯或L - 核糖核苷酸后的3',5'-磷酸二酯的DNA酶。这两种酶都能以多轮周转的分子间反应形式发挥作用。切割2',5'-磷酸二酯的DNA酶表现出约0.01 min⁻¹的kcat和大约10⁸ M⁻¹ min⁻¹的催化效率kcat/Km。切割L - 核糖核苷酸的酶速度约慢10倍，催化效率约为4×10⁵ M⁻¹ min⁻¹。两种酶的活性都需要二价金属阳离子，并且在pH 7 - 8和35 - 50℃时具有最佳催化速率。在比较每种酶对含有非天然核糖核苷酸的相应底物或含有标准核糖核苷酸的底物的活性时，切割2',5'-磷酸二酯的DNA酶表现出6000倍的区域选择性，而切割L - 核糖核苷酸的DNA酶表现出40倍的对映选择性。这些分子展示了体外进化如何用于获得对生物化合物的非天然类似物具有特异性的区域和对映选择性催化剂。
