INHIBITION OF DIHYDROFOLATE-REDUCTASE - EFFECT OF REDUCED NICOTINAMIDE ADENINE-DINUCLEOTIDE PHOSPHATE ON THE SELECTIVITY AND AFFINITY OF DIAMINOBENZYLPYRIMIDINES
INHIBITION OF DIHYDROFOLATE-REDUCTASE - EFFECT OF REDUCED NICOTINAMIDE ADENINE-DINUCLEOTIDE PHOSPHATE ON THE SELECTIVITY AND AFFINITY OF DIAMINOBENZYLPYRIMIDINES
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DOI：
10.1021/bi00263a034
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发表时间：
1982-01-01
期刊：
影响因子：
2.9
通讯作者：
KING, RW
中科院分区：
文献类型：
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作者：
BACCANARI, DP;DALUGE, S;KING, RW
用平衡法和动力学方法研究了苄基嘧啶类化合物对细菌二氢叶酸还原酶的选择性。甲氧苄啶[2，4-二氨基-5-（3，4，5-三甲氧基苄基）嘧啶]和一系列苄基上甲氧基取代不同的结构类似物显示出不同取代度的体外大肠杆菌抗菌活性。甲氧苄啶，最有效的类似物测试，是400倍以上的活性比苄基嘧啶，单甲氧基和二甲氧基类似物的中间抗菌活性。所有化合物的相对抗菌活性均与其大肠杆菌数成正比。大肠杆菌1型二氢叶酸还原酶Ki值，如通过经典酶动力学测定的。酶测定的抑制是酶-抑制剂-NADPH三元复合物的量度，Ki值范围为670 nM（未取代的苄基嘧啶）至1.3 nM（甲氧苄啶）。用E.大肠杆菌和干酪乳杆菌的酶在NADPH的情况下进行的研究表明，从酶抑制剂二元复合物的抑制剂的解离没有广泛的动力学Ki值的变化，这些二元常数不直接相关的动力学Ki或抗菌活性。NADPH增加了细菌酶对抑制剂的亲和力;酶-抑制剂-NADPH三元复合物中这种增加的亲和力（协同性）随甲氧基取代的程度（≥）而变化。230-甲氧苄氨嘧啶和2型E.大肠杆菌酶）。与此相反，所有化合物都是哺乳动物酶（SR-1啮齿动物淋巴瘤）的弱抑制剂，并且没有一种化合物显示出与NADPH超过8倍的结合协同性。因此，NADPH协同性是大肠杆菌高亲和力的重要因素。大肠杆菌二氢叶酸还原酶对甲氧苄啶的作用以及与哺乳动物酶的协同性的缺乏在甲氧苄啶作为抗菌剂的选择性中是重要的。
The selectivity of benzylpyrimidines for bacterial dihydrofolate reductases was studied by equilibrium and kinetic techniques. Trimethoprim [2,4-diamino-5-(3,4,5-trimethoxybenzyl)pyrimidine] and a series of close structural analogs with different methoxy group substituions on the benzyl showed in vitro Escherichia coli antibacterial activities that varied according to their degree of substitution. Trimethoprim, the most potent analog tested, was 400-fold more active than benzylpyrimidine; the monomethoxy and dimethoxy analogs were of intermediate antibacaterial activity. The relative antibacterial potencies of all the compounds were directly proportional to their E. coli form 1 dihydrofolate reductase Ki values, as determined by classical enzyme kinetics. Inhibition of the enzyme assay is a measure of the enzyme-inhibitor-NADPH ternary complex and the Ki values ranged from 670 nM for the unsubstituted benzylpyrimidine to 1.3 nM for trimethoprim. Equilibrium dialysis and fluorescence studies with the E. coli and Lactobacillus casei enzymes performed in the absence of NADPH showed that the dissociation of inhibitors from the enzyme-inhibitor binary complex did not vary as widely as kinetic Ki values and that these binary constants were not directly related to kinetic Ki or antibacterial activity. NADPH increased the affinity of the bacterial enzymes for inhibitors; this increased affinity in the enzyme-inhibitor-NADPH ternary complex (cooperativity) varied with the degree of methoxy substitution (.gtoreq. 230-fold for trimethoprim and the form 2 E. coli enzyme). Contrary to this, all the compounds were weak inhibitors of the mammalian enzyme (SR-1 rodent lymphoma) and none showed more than 8-fold binding cooperativity with NADPH. Therefore, NADPH cooperativity is an important factor in the high affinity of E. coli dihydrofolate reductase for trimethoprim and the lack of cooperativity with the mammalian enzyme is important in the selectivity of trimethoprim as an antibacterial.