Neumann P, Markau K, Sund H
Eur J Biochem. 1976 Jun 1;65(2):465-72. doi: 10.1111/j.1432-1033.1976.tb10362.x.
Glutamate dehydrogenase from Candida utilis undergoes a reversible conformational transition between an active and an inactive state at low pH AND low temperature. This conformational transition can also be followed by fluorescence measurements. The temperature-dependent equilibrium between the active and the inactive state is characterized by a transition temperature of 10.7 degrees C and a delta H value of 148 kcal/mol (620 kJ/mol). The temperature dependence of the enzymic activity above 15 degrees C yields an activation energy of 15 kcal/mol (63 kJ/mol), a larger value than that for the beef liver enzyme (9 kcal/mol; 38 kJ/mol). In contrast to the yeast enzyme the Arrhenius plot is linear and, therefore, the beef liver enzyme is not transformed into an inactive conformation at low temperatures. Sedimentation analysis shows that the inactivation of the Candida utilis enzyme is not caused by change in the quaternary structure. The pH dependence of the conformational transition at low pH measured by fluorescence change is characterized by a pK value of 7.01 for the enzyme in the absence and of 6.89 for the enzyme in the presence of 2-oxoglutarate with a Hill coefficient of 3.4 in both cases. Similar results are found when the pH dependence of the enzymic activity is analyzed. With the beef liver enzyme the same pK value is obtained but with a Hill coefficient of 1 indicating cooperativity only in the case of the Candida utilis enzyme. The best fit of the pH dependence of the rate constants of the fluorescence changes was obtained with pK values of 7.45 and 6.45 for the active and the inactive state respectively. In this model the lowest time constant which is obtained at the pH of the equilibrium was found to be 0.05 s-1. Preincubation experiments with the substrate 2-oxoglutarate but not with the coenzyme shift the equilibrium to the active conformation. The coenzyme obviously reduces the rate constant of the conformational transition. The sedimentation coefficient (SO20, w) and the molecular weight were found to be 11.0 S and 276 000, respectively. The enzyme molecule is built up by six polypeptide chains each having a molecular weight of 47 000.
产朊假丝酵母的谷氨酸脱氢酶在低pH值和低温条件下会在活性状态和非活性状态之间发生可逆的构象转变。这种构象转变也可以通过荧光测量来跟踪。活性状态和非活性状态之间的温度依赖性平衡的特征是转变温度为10.7℃，ΔH值为148千卡/摩尔（620千焦/摩尔）。15℃以上酶活性的温度依赖性产生的活化能为15千卡/摩尔（63千焦/摩尔），该值大于牛肝酶的活化能（9千卡/摩尔；38千焦/摩尔）。与酵母酶不同，牛肝酶的阿累尼乌斯图是线性的，因此，牛肝酶在低温下不会转变为非活性构象。沉降分析表明，产朊假丝酵母酶的失活不是由四级结构的变化引起的。通过荧光变化测量的低pH值下构象转变的pH依赖性，在不存在2-氧代戊二酸时，酶的pK值为7.01，在存在2-氧代戊二酸时，酶的pK值为6.89，两种情况下的希尔系数均为3.4。分析酶活性的pH依赖性时也发现了类似结果。对于牛肝酶，获得了相同的pK值，但希尔系数为1，表明只有产朊假丝酵母酶存在协同作用。荧光变化速率常数的pH依赖性的最佳拟合分别得到活性状态和非活性状态的pK值为7.45和6.45。在该模型中，在平衡pH值下获得的最低时间常数为0.05 s-1。用底物2-氧代戊二酸而不是辅酶进行预孵育实验会使平衡向活性构象移动。辅酶明显降低了构象转变的速率常数。沉降系数（S20,w）和分子量分别为11.0 S和276 000。酶分子由六条多肽链组成，每条多肽链的分子量为47 000。
