Nardone G, George J, Chirikjian J G
J Biol Chem. 1984 Aug 25;259(16):10357-62.
BamHI methylase has been purified to apparent homogeneity. The isolated form of the enzyme is a single polypeptide with a molecular weight of 56,000 as determined by sodium dodecyl sulfate-polyacrylamide electrophoresis. Unlike BamHI endonuclease, which is isolated as a dimer and higher aggregates, the methylase has no apparent higher form. The methylase requires S-adenosyl-L-methionine as the methyl-group donor and is inhibited by Mg2+. The enzyme is also inhibited by 2,3-butanedione and reagents specific for sulfhydryl groups, such as N-ethylmaleimide, which suggests a role for arginine and cysteine residues, respectively. DNA efficiently protects the enzyme against the butanedione modification while S-adenosylmethionine has no effect. In contrast, S-adenosylmethionine protects against cysteine modification while DNA produces only small amounts of protection. Studies on the mechanism of methylation indicate that both strands of the recognition sequence are modified in a single binding event. The sequence specificity of the methylase is relaxed upon the addition of glycerol in the reaction mixture. In the presence of 30% glycerol the enzyme methylates sequences that are also recognized by BamHI endonuclease when acting under conditions of relaxed specificity.
BamHI甲基化酶已被纯化至表观均一。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳测定，该酶的分离形式为单一多肽，分子量为56,000。与以二聚体和更高聚集体形式分离的BamHI核酸内切酶不同，甲基化酶没有明显的更高形式。甲基化酶需要S-腺苷-L-甲硫氨酸作为甲基供体，并受到Mg2+的抑制。该酶也受到2,3-丁二酮和巯基特异性试剂（如N-乙基马来酰亚胺）的抑制，这分别表明精氨酸和半胱氨酸残基发挥了作用。DNA能有效保护该酶免受丁二酮修饰，而S-腺苷甲硫氨酸则无作用。相反，S-腺苷甲硫氨酸能保护免受半胱氨酸修饰，而DNA仅产生少量保护作用。甲基化机制研究表明，识别序列的两条链在单次结合事件中均被修饰。在反应混合物中加入甘油后，甲基化酶的序列特异性会放宽。在30%甘油存在下，当在特异性放宽的条件下起作用时，该酶会甲基化BamHI核酸内切酶也能识别的序列。
