Sibirtsev Y T, Rasskazov V A
Pacific Institute of Bioorganic Chemistry, Far East Branch of the Russian Academy of Sciences, Vladivostok, 690022, Russia.
Biochemistry (Mosc). 2000 Aug;65(8):952-8.
The effect of ionic strength of the reaction medium on the pH optimum, specificity, and mechanism of action of the acid DNase isolated from mature eggs of the sea urchin Strongylocentrotus intermedius was studied. Changes in ionic strength of the reaction medium caused a displacement of the pH optimum of the enzyme to acidity or alkalinity. The region and range of this displacement depended on the buffer used and on the substrate structure. For single-stranded, duplex, and supercoiled forms of DNA, the pH optimum displacements were 1.0, 1.4, and 2.0 pH units, respectively. The pH optimum displacement changed the mechanism of action of the enzyme. Under optimum pH conditions, the enzyme cleaved supercoiled DNA only by the double-hit mechanism, and fragments of duplex DNA resulted due to the coincidence of breaks in opposite chains. On pH displacement to acidity, the enzyme acted by the mixed mechanism (single- and double-hit). And the quantitative ratio of products of the enzymatic hydrolysis of supercoiled DNA was significantly changed depending on the pH displacement to acidity or to alkalinity. The findings are explained by the effect of salt-dependent electrostatic interactions during the formation of a nonspecific DNA-enzyme complex.
研究了反应介质的离子强度对从中间球海胆成熟卵中分离出的酸性脱氧核糖核酸酶（DNase）的最适pH值、特异性及作用机制的影响。反应介质离子强度的变化导致酶的最适pH值向酸性或碱性方向偏移。这种偏移的范围和区域取决于所使用的缓冲液及底物结构。对于单链、双链和超螺旋形式的DNA，最适pH值的偏移分别为1.0、1.4和2.0个pH单位。最适pH值的偏移改变了酶的作用机制。在最适pH条件下，该酶仅通过双击机制切割超螺旋DNA，双链DNA片段是由于相对链上的断裂重合产生的。当pH值向酸性偏移时，酶通过混合机制（单击和双击）起作用。并且超螺旋DNA酶促水解产物的定量比例会因pH值向酸性或碱性的偏移而发生显著变化。这些发现可以通过非特异性DNA - 酶复合物形成过程中盐依赖性静电相互作用的影响来解释。
