Gilbert H J, Drabble W T
Biochem J. 1980 Nov 1;191(2):533-41. doi: 10.1042/bj1910533.
IMP dehydrogenase of Escherichia coli was irreversibly inactivated by Cl-IMP (6-chloro-9-beta-d-ribofuranosylpurine 5'-phosphate, 6-chloropurine ribotide). The inactivation reaction showed saturation kinetics. 6-Chloropurine riboside did not inactivate the enzyme. Inactivation by Cl-IMP was retarded by ligands that bind at the IMP-binding site. Their effectiveness was IMP>XMP>GMP>>AMP. NAD(+) did not protect the enzyme from modification. Inactivation of IMP dehydrogenase was accompanied by a change in lambda(max.) of Cl-IMP from 263 to 290nm, indicating formation of a 6-alkylmercaptopurine nucleotide. The spectrum of 6-chloropurine riboside was not changed by IMP dehydrogenase. With excess Cl-IMP the increase in A(290) with time was first-order. Thus it appears that Cl-IMP reacts with only one species of thiol at the IMP-binding site of the enzyme: 2-3mol of Cl-IMP were bound per mol of IMP dehydrogenase tetramer. Of ten mutant enzymes from guaB strains, six reacted with Cl-IMP at a rate similar to that for the native enzyme. The interaction was retarded by IMP. None of the mutant enzymes reacted with 6-chloropurine riboside. 5,5'-Dithiobis-(2-nitrobenzoic acid), iodoacetate, iodoacetamide and methyl methanethiosulphonate also inactivated IMP dehydrogenase. Reduced glutathione re-activated the methanethiolated enzyme, and 2-mercaptoethanol re-activated the enzyme modified by Cl-IMP. IMP did not affect the rate of re-activation of methanethiolated enzyme. Protective modification indicates that Cl-IMP, methyl methanethiosulphonate and iodoacetamide react with the same thiol groups in the enzyme. This is also suggested by the low incorporation of iodo[(14)C]acetamide into Cl-IMP-modified enzyme. Hydrolysis of enzyme inactivated by iodo[(14)C]acetamide revealed radioactivity only in S-carboxymethylcysteine. The use of Cl-IMP as a probe for the IMP-binding site of enzymes from guaB mutants is discussed, together with the possible function of the essential thiol groups.
大肠杆菌的肌苷酸脱氢酶被Cl-IMP（6-氯-9-β-D-呋喃核糖基嘌呤5'-磷酸，6-氯嘌呤核苷酸）不可逆地失活。失活反应呈现饱和动力学。6-氯嘌呤核苷不会使该酶失活。在IMP结合位点结合的配体可延缓Cl-IMP导致的失活。它们的效力顺序为IMP>XMP>GMP>>AMP。NAD(+)不能保护该酶免受修饰。肌苷酸脱氢酶失活伴随着Cl-IMP的最大吸收波长从263nm变为290nm，这表明形成了6-烷基巯基嘌呤核苷酸。6-氯嘌呤核苷的光谱未被肌苷酸脱氢酶改变。在过量Cl-IMP存在下，A(290)随时间的增加呈一级反应。因此，似乎Cl-IMP仅与该酶IMP结合位点的一种硫醇反应：每摩尔肌苷酸脱氢酶四聚体结合2 - 3摩尔Cl-IMP。来自guaB菌株的十种突变酶中，六种与Cl-IMP反应的速率与天然酶相似。IMP可延缓这种相互作用。没有一种突变酶与6-氯嘌呤核苷反应。5,5'-二硫代双-(2-硝基苯甲酸)、碘乙酸、碘乙酰胺和甲基甲硫基磺酸盐也能使肌苷酸脱氢酶失活。还原型谷胱甘肽可使甲硫基化的酶重新激活，2-巯基乙醇可使被Cl-IMP修饰的酶重新激活。IMP不影响甲硫基化酶的重新激活速率。保护性修饰表明Cl-IMP、甲基甲硫基磺酸盐和碘乙酰胺与该酶中相同的硫醇基团反应。碘代[(14)C]乙酰胺掺入Cl-IMP修饰酶中的量较低也表明了这一点。对碘代[(14)C]乙酰胺失活的酶进行水解后，放射性仅出现在S-羧甲基半胱氨酸中。本文讨论了使用Cl-IMP作为guaB突变体酶IMP结合位点的探针，以及必需硫醇基团的可能功能。
