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测定中华仓鼠二氢叶酸还原酶（ＤＨＦＲ，Ｅ．Ｃ．１．５．１．３．）催化反应的各个动力学常数，对其反应机制进行了研究。测定了不同浓度脲溶液中酶与底物的解离常数Ｋｄ和表观米氏常数Ｋｍ，结果表明酶与底物二氢叶酸（ＤＨＦ）和还原型尼克酰胺腺嘌呤二核苷酸磷酸（ＮＡＤＰＨ）的结合能力相差很小，都随脲浓度增加而减弱，而且一种底物的结合会削弱酶与另一底物的结合能力。研究了ＤＨＦＲ在脲变性过程中活力和构象的变化，结果表明低浓度脲可使稳态酶活力增强，此时酶的内源荧光发射光谱和ＣＤ谱变化很小；随脲浓度的增加，酶逐渐失活，同时荧光光谱的最大发射峰位红移，荧光强度和椭圆率也明显下降，说明酶活力的变化先于酶分子整体构象的变化，酶活性部位位于比酶分子整体更易受变性剂影响的有限区域，而且酶分子这种相对的和一定限度内的柔性是其表现生物活性所必需的
The kinetic constants of the catalytic reaction of DHFR (DHFR, E.C.1.5.1.3.) Were determined and the reaction mechanism was studied. The dissociation constants (Kd) and apparent Michaelis constant (Km) of enzyme and substrate in different concentrations of urea solution were determined. The results showed that the interaction between enzyme and substrate DHF and NADPH ) Have little difference in binding ability, both decrease with the increase of urea concentration, and the binding of one kind of substrate will weaken the binding ability of enzyme to another substrate. The changes of viability and conformation of DHFR during urea denaturation were studied. The results showed that the activity of steady-state enzyme increased with low concentration of urea, and the change of endogenous fluorescence emission spectrum and CD spectrum of enzyme was small. With the increase of urea concentration, While the peak of fluorescence peak red-shifted, the fluorescence intensity and ellipticity also decreased significantly, indicating that the change of enzyme activity preceded the change of the overall conformation of the enzyme molecule, and the active site of the enzyme was more susceptible to the denaturant Limited area of influence, and this relative and limited flexibility of the enzyme molecule is necessary for its biological activity