Bam HI DNA甲基化酶酶学性质的研究

Enzymology of Ban HI DNA Methylase

以Lineweave-Burk plot双倒数作图法测得该酶对底物S-腺苷酰甲硫氨酸(SAM)的K_m=7.69×10^(-6)mol/L,在1mmol/LS-腺苷酰高半胱氨酸(SAH)存在下,Ki=7.33×10^(-4)mol/L,两条直线相交于纵轴,证明SAH是该酶的竞争性抑制剂。该酶最适pH为7.8,对热不稳定。同时还测定了该酶对不同DNA底物的专一性及盐浓度、代谢相关物’两价阳离子、某些酸根等对该酶调节性质的影响。以碘代乙酰胺修饰该酶的SH基’及用二硫苏糖醇(DTT)和巯基乙醇(MSH)保护该酶SH基所作的实验表明SH基是该酶活性中心所必需的,用高效液相色谱(HPLC)法证明该酶所甲基化的碱基为刘氏小球菌(M·L、DNA)分子中的胞嘧啶,且求得甲基化30min后所得甲基化水平为2.39％。同时也证明当用该酶将λDNA甲基化后,可使BamHI限制性核酸内切酶对甲基化后的λDNA丧失切割作用。

The enzymology of Bam HI DNA methylase has been studied.The optimum pH is 7.8.It is unstable in heat treatment and dependent on K+ or Na+. The Km is 7.69 × 10-6mol/L for SAM and the Ki is 7.33 × 10-4mol/L in the presence of 1 mmol/L SAH by L-B plot.The two lines intersected at the ordinate on L-B plot. This re sult shows that SAH is the competitive inhibitor for Bam HI DNA methylase. The study of substrate specificity for different DNA shows that the single stranded M.L. DNA is the best substrate for this enzyme.The enzyme activity was assayed in the presence of different metabolites such as cytosine, 5-azacytosine, adenosine, 6 -azacytosine, 5 -azacytidine and 5-methylcy-tosine.The enzyme activity could be inhibited by divalent cations such as Mn2+,Mg2+, Zn2+, but was stimulated strongly by F-, WO43-, MoO42-.Results from the chemical modification by iodoacetamide and protection by DTT or 2 -mercaptoethanol of this enzyme showed that the SH group is essential in active site of this enzyme.It has been proved by HPLC that this enzyme transfred the methyl group from SAM to cyto sine of DNA.The methylation level of M.L.DNA is 2.39% when it was incubated with Bam HI DNA methylase for 30'.It has been proved by electrophoresis on 1% agarose that the Bam HI DNA methylase modified DNA can not be cleaved by Bam HI restriction endonuclease.