Studies of the role of catalytic and conformational metals in producing enzymatic activity in yeast enolase.
Studies of the role of catalytic and conformational metals in producing enzymatic activity in yeast enolase.
复制标题
研究催化和构象金属在酵母烯醇酶产生酶活性中的作用。
DOI：
10.1016/s0162-0134(00)80118-x
复制
发表时间：
1980
影响因子：
3.9
通讯作者：
K. M. Collins
中科院分区：
文献类型：
--
作者：
J. Brewer;K. M. Collins
分光光度滴定的酵母脱辅烯醇酶与镁，金属产生的最高水平的活动，镍，产生一个非常低的水平，和钙，它不产生任何活动，建议强结合的2摩尔（1每个亚基）的所有三种金属在相同的网站，称为“构象”的网站。当仅加入2 mol（1/亚基）镁时，在饱和水平的镁存在下与酶结合时，发色团竞争性抑制剂3-氨基烯醇丙酮酸-2-磷酸（AEP）中可能发生的吸光度变化的约三分之二产生。由于额外的“催化”金属不会结合，除非AEP结合，而AEP不会结合，除非“构象”位点被金属填充，AEP中的大部分吸光度变化必须由构象金属产生。不产生酶活性的金属不产生AEP中的吸光度变化，而允许任何水平的酶活性的金属产生与镁相同的吸光度变化。反应是“全有或全无”。钙，镍，镁的CD光谱的apoenzyme AEP解决方案的影响的研究表明，激活金属产生的AEP中的不对称发色团。量热法研究表明，竞争性抑制剂3-磷酸甘油酸与钙、镁酶结合的焓变基本相同。底物或AEP降低了钙或镁酶与过量EDTA的表观反应速率，表明底物和AEP都与钙酶结合。对这些数据的解释是，构象金属在活化底物中起着至关重要的作用，而催化金属控制反应速率。这种解释是支持的实验中，一种类型的构象金属的酶与催化金属和底物的停流反应。如果活化金属是构象金属，则初始活性更大。
Spectrophotometric titrations of yeast apoenolase with magnesium, the metal that produces the highest level of activity, nickel, which produces a very low level, and calcium, which produces no activity, suggest strong binding of 2 mol (1 per subunit) of all three metals at the same sites, called “conformational” sites. About two-thirds of the possible absorbance change in the chromophoric competitive inhibitor 3-aminoenolpyruvate-2-phosphate (AEP) that occurs when it binds to the enzyme in the presence of saturating levels of magnesium is produced when just 2 mol (1 per subunit) of magnesium is added. Since additional “catalytic” metal won't bind unless the AEP does, and the AEP won't bind unless the “conformational” sites are filled with metal, much of the absorbance change in the AEP must be produced by conformational metal.Metals that do not produce enzymatic activity do not produce the absorbance change in AEP whereas metals that permit any level of enzymatic activity produce the same absorbance change that magnesium does-the reaction is “all or none.” Studies of the effect of calcium, nickel, and magnesium on the CD spectrum of apoenzyme-AEP solutions suggest that activating metals produce an asymmetric chromophore in the AEP. This is interprested as indicating the chromophore in AEP bound to enzyme in the presence of an activating metal is a twisted carbon-carbon double bond.Calorimetric studies show the competitive inhibitor 3-phosphoglycerate binds to the calcium- and magnesium-enzyme with about the same change in enthalpy. The substrate or AEP reduces the rate of the apparent reaction of the calcium- or magnesium-enzyme with excess EDTA, suggesting that both substrate and AEP bind to the calcium-enzyme. The interpretation of these data is that the conformational metal plays a crucial role in activating the substrate while the catalytic metal controls the reaction rate. This interpretation is supported by experiments in which an enzyme with one type of conformational metal is reacted in the stopped-flow with catalytic metal and substrate. If an activating metal is the conformational metal, the initial activity is greater.