COENZYME RECOGNITION BY RAT VITAMIN B12-DEPENDENT METHIONINE SYNTHASE

由于哺乳动物组织中含有极低水平的蛋氨酸合成酶，我们建立了一种利用杆状病毒表达系统在昆虫细胞中高效表达大鼠蛋氨酸合成酶基因的方法。表达的酶主要是脱辅酶，很不稳定。与甲钴胺络合后，功能全酶得到纯化。底物的比活力和表观K_m值与用纯化的大鼠肝酶测定的结果一致。纯化后的重组酶的电子光谱与CoB(II)-丙氨酸钴相似，但与钛(III)和S-腺苷甲硫氨酸孵育后变为类甲钴胺。核苷酸部分，特别是磷酸二酯基，在辅酶与脱辅基蛋白的结合中起着重要的作用，从而起到催化作用。在没有还原剂的情况下与脱氢酶孵育时，氰基和水钴胺对…无效。在重组全酶方面，r更有效，但效果不明显。乙基钴胺和丙基钴胺与脱辅酶形成失活的络合物，通过光解活化转化为全酶。腺苷钴胺不能与酶形成复合体。用B_(12)缺乏饲料饲养产生了严重的B_(12)缺乏大鼠。尿甲丙二酸排泄量增加，肝脏B_12&gt；含量和钴胺依赖蛋氨酸合成酶活性降低，证实了B_&lt；12&gt；-缺乏的状态。研究表明，大鼠肝脏蛋氨酸合成酶几乎以全酶形式存在。当B_(12)缺乏时，蛋氨酸合成酶蛋白水平下降，而mRNA水平没有变化。当给予B_(12)缺陷大鼠甲钴胺时，生长、肝脏B_(12)水平和尿甲基丙二酸甲酯排泄量恢复到对照水平(B_>充足)。在此恢复过程中，蛋氨酸合成酶活性和蛋白水平上升，而mRNA水平基本保持不变。我们在上面已经提到，大鼠去甲硫氨酸合成酶是非常不稳定的，并且通过与甲钴胺的络合而显著稳定。因此，在B&lt；12&gt；缺陷动物中，哺乳动物蛋氨酸合成酶活性的降低可以解释为不是“辅酶诱导”，而是通过钴胺结合稳定酶(“辅酶稳定”)。较少

Since the mammalian tissues contain the enzyme in an extremely low level, we established a method for high-level expression of rat methionine synthase cDNA in insect cells using a baculovirus expression system. The expressed enzyme was chiefly as apoenzyme and very unstable. After complexation with methylcobalamin, the functional holoenzyme was purified to homogeneity. The specific activity and apparent K_m values for substrates were in good agreement with those obtained with purified rat liver enzyme. The electronic spectrum of the purified recombinant enzyme resembled that of cob (II) alamin and changed to a methylcobalamin-like one upon incubation of the enzyme with titanium (III) and S-adenosylmethionine. The nucleotide moiety, especially the phosphodiester group, was shown to play an important role in the binding of the coenzyme to apoprotein and thus for catalysis. Upon incubation with the apoenzyme in the absence of a reducing agent, cyano- and aquacobalamin were not effective o … More r effective only slightly in reconstituting holoenzyme. Ethyl- and propylcobalamin formed inactive complexes with apoenzyme, which were converted to holoenzyme by photolytic activation. Adenosylcobalamin was not able to form a complex with apoenzyme.Severe B_<12>-deficient rats were produced by breeding with B_<12>-deficient diet. The status of B_<12>-deficiency was confirmed by increase in urinary methylmalonate excretion and decreases in liver B_<12> contents and cobalamin-dependent methionine synthase activity. It was demonstrated that rat liver methionine synthase exists almost exclusively as holoenzyme. Upon B_<12>-deficiency, the level of methionine synthase protein decreased, whereas the mRNA level did not change. When methylcobalamin was administered to the B_<12>-deficient rats, growth, liver B_<12> levels, and urinary excretion of methylmalonate reversed to the control (B_<12>-sufficient) levels. During this recovery process, methionine synthase activity and protein levels increased, while the mRNA level remained essentially unchanged. We have mentioned above that rat apomethionine synthase is very unstable and markedly stabilized by complexation with methylcobalamin. Thus, the decrease of mammalian methionine synthase activity in B_<12>-deficient animals can be interpreted in terms of not "coenzyme induction" but stabilization of the enzyme by cobalamin binding ("coenzyme stabilization"). Less