Binding of methotrexate to dihydrofolate reductase and its relation to protonation of the ligand.
Binding of methotrexate to dihydrofolate reductase and its relation to protonation of the ligand.
复制标题
甲氨蝶呤与二氢叶酸还原酶的结合及其与配体质子化的关系。
DOI：
10.1021/bi00338a032
复制
发表时间：
1985
期刊：
影响因子：
2.9
通讯作者：
Cocco,L
中科院分区：
文献类型：
--
作者：
Blakley,RL;Cocco,L
Revised Manuscript Received March 18, 1985 abstract: Stopped-flow spectrophotometry and stopped-flow fluorometry havebeen used tostudy the binding of methotrexate (MTX) and 3-deazamethotrexate (3-deazaMTX) to dihydrofolate reductase (DHFR) isoenzymes from Streptococcus faecium and from Lactobacillus casei. The absorbance change and fluorescence quenching that occur when MTX binds to DHFR isoenzyme II from S. faecium (SFDHFR II) are both biphasic and give similar apparent rate constants for both phases. The faster phase has an apparent rate constant that is dependent on MTX concentration and therefore corresponds to the initial binding reaction. From the concentration dependence it has been calculated that the association rate constant is 3.0 X 105 M"* 1 s-1 at 20 C and pH 7.3, and the association constant (equilibrium constant) under these conditions is 5.8 X 105 M-1. By examination of the amplitude of the fast-phase absorbance change at various wavelengths, it has been determined that the absorbance change occurring in the fast phase is due to MTX protonation. Within the limits of the method it was thus not possible to detect a difference in therates of binding and of protonation of MTX. The MTX association rate constant is pH dependent, decreasing 330-fold as the pH is decreased from 5.0 to 9.0. The data fit well to a curve generated by assuming a single ionization with a pATa of 6.0 and a pH-independent association rate constant 1000-fold greater for binding of protonated MTX to SFDHFR II than for binding of unprotonated MTX. In the case of 3-deazaMTX binding the data fit to a curve corresponding to a pKa of 7.5 and 200-fold higher pH-independent rate constant for the protonated species than for the unprotonated. Binding of MTX to isoenzyme I of S. faecium DHFR and to DHFR fromL. casei occurs with association and dissociation rate constants quitesimilar to those for binding to SFDHFR II. However, the binding of the protonatedform of MTX is favored by a factor of only 200-fold in the case of S. faecium DHFR isoenzyme I and only7-fold in the case of L. casei DHFR, despite the fact that the overall associationconstants for the initial binding ofMTX to the three types of DHFR are comparable. Thus, the relative rates of binding of protonated MTX and unprotonated MTX to DHFR vary with the source of the enzyme and hence its detailed structure. Thermodynamic association constants determined by fluorescent titration are significantly higher than those calculatedfrom ratios of rate constants for association and dissociation of complexes. This implies occurrence of important isom-erizations of the complexes after the initial binding reaction. The data also suggest that the protonated ligand-active site carboxylateinteraction has a limited effect on the rate of inhibitor binding in the case of MTX binding to DHFR from L. casei and to isoenzyme I from S. faecium, though it greatly increases the binding rate in the case of SFDHFR II. However, even in the latter case the ion pair interaction does not result in an unusually high associationconstant for the initial binding reaction.Dihydrofolate reductase (5, 6, 7, 8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5. 1.3; DHFR) 1 catalyzes the NADPH-dependent reduction of H2folate to H4fplate. The metabolic importance of ÜHFR, which stems from the fact that deriv-atives of its product, H4folate, have coenzyme functions in one-carbon metabolism, has inspired extensive research on inhibitors of the reductase, several of which are widely used
