Liao T H, Hsieh J C
Department of Biochemistry, National Taiwan University/College of Medicine, Taipei, Republic of China.
Biochem J. 1988 Nov 1;255(3):781-7. doi: 10.1042/bj2550781.
The ability of bovine pancreatic DNAase to hydrolyse the synthetic substrate p-nitrophenyl phenylphosphonate (NPPP) is intrinsic and is not due to the contamination of the DNAase preparation by nonspecific phosphodiesterases because the activities of DNA and NPPP hydrolysis are co-eluted from a DEAE-cellulose column with use of the Ca2+-affinity elution method and because the two activities are decreased simultaneously when the purified enzyme is treated with Cu2+/iodoacetate, an active-site-labelling agent for DNAase. NPPP hydrolysis is facilitated by the metal ion-DNAase. At relatively high Na+ concentrations, where the metal ion-DNA interaction is weak, DNA hydrolysis is also facilitated by the metal ion-DNAase. With NPPP as substrate the Michaelis constants are Km 3.7 mM for Mn2+ and Km 49 mM for Mg2+ in 0.2 M-Tris/HCl buffer, pH 7.2. Ca2+ competes with Mn2+, with Ki 64 mM. Free Cu2+ ions non-competitively inhibit DNAase-catalysed DNA or NPPP hydrolysis in the presence of Mn2+ or Mg2+ and the inhibition is not relieved by Ca2+. The affinity of Cu2+ for free DNAase is higher than that for Mn2+-DNAase. Mn2+ is not bound to DNAase via a simple ionic interaction, as Mn2+ remains bound in the presence of relatively high Na+ concentrations and induces a near-u.v. difference absorption spectrum. The kinetics of NPPP hydrolysis catalysed by Mn2+-DNAase are sigmoidal. From the Hill equation, h = 2.0 is obtained, suggesting that more than two NPPP molecules are bound per molecule of DNAase with a certain amount of co-operativity. Because DNAase in solution is a monomer with a single catalytic site, the multiple NPPP molecules on a single protein molecule are probably in one location, resulting in a co-operative interaction that may resemble that in the stacked base-pairs of double-helical DNA.
牛胰脱氧核糖核酸酶水解合成底物对硝基苯基苯基膦酸酯（NPPP）的能力是其固有的，并非由于该脱氧核糖核酸酶制剂被非特异性磷酸二酯酶污染，这是因为采用Ca2+亲和洗脱法时，DNA水解活性和NPPP水解活性从DEAE - 纤维素柱上共同洗脱，还因为当用脱氧核糖核酸酶的活性位点标记剂Cu2+/碘乙酸处理纯化的酶时，这两种活性会同时降低。金属离子 - 脱氧核糖核酸酶可促进NPPP的水解。在相对较高的Na+浓度下，金属离子与DNA的相互作用较弱，金属离子 - 脱氧核糖核酸酶也能促进DNA的水解。以NPPP为底物时，在0.2 M - Tris/HCl缓冲液（pH 7.2）中，Mn2+的米氏常数Km为3.7 mM，Mg2+的Km为49 mM。Ca2+与Mn2+竞争，抑制常数Ki为64 mM。在存在Mn2+或Mg2+的情况下，游离的Cu2+离子非竞争性抑制脱氧核糖核酸酶催化的DNA或NPPP水解，且Ca2+不能解除这种抑制。Cu2+对游离脱氧核糖核酸酶的亲和力高于对Mn2+ - 脱氧核糖核酸酶的亲和力。Mn2+并非通过简单的离子相互作用与脱氧核糖核酸酶结合，因为在相对较高的Na+浓度下Mn2+仍保持结合状态，并诱导出近紫外差示吸收光谱。由Mn2+ - 脱氧核糖核酸酶催化的NPPP水解动力学呈S形。根据希尔方程，得到h = 2.0，这表明每分子脱氧核糖核酸酶结合的NPPP分子超过两个，存在一定程度的协同性。由于溶液中的脱氧核糖核酸酶是具有单个催化位点的单体，单个蛋白质分子上的多个NPPP分子可能位于同一位置，从而导致协同相互作用，这种相互作用可能类似于双螺旋DNA堆积碱基对中的相互作用。
