Huang Y C, Colman R F
J Biol Chem. 1984 Oct 25;259(20):12481-8.
Pig heart NAD-dependent isocitrate dehydrogenase is allosterically activated by ADP which reduces the Km of isocitrate. The new ADP analogue 6-(4-bromo-2,3-dioxobutyl)thioadenosine 5'-diphosphate (BDB-TADP) reacts irreversibly with the enzyme at pH 6.1 and 25 degrees C, causing a rapid loss of the ability of ADP to increase the initial velocity of assays conducted at low isocitrate concentrations and a slower inactivation measured using saturating isocitrate concentrations. The rate constant for loss of ADP activation exhibits a nonlinear dependence on BDB-TADP concentration; in the presence of 0.2 mM MnSO4, KI for the reversible enzyme-reagent complex is 0.069 mM with kmax at saturating reagent concentrations equal to 0.031 min-1. For reaction at the site causing overall inactivation, KI for the initial reversible enzyme-reagent complex is estimated to be 0.018 mM with kmax = 0.0083 min-1 in the presence of 0.2 mM MnSO4. Total protection against both reactions is provided by 1 mM ADP plus 0.2 mM MnSO4 or by 0.1 mM ADP plus 0.2 mM MnSO4 plus 0.2 mM isocitrate, but not by NAD, ATP, or ADP plus EDTA. The BDB-TADP thus appears to modify two distinct metal-dependent ADP-binding sites. Incubation of isocitrate dehydrogenase with 0.14 mM BDB-[beta-32P]TADP at pH 6.1 in the presence of 0.2 mM MnSO4 results in incorporation of 0.81 mol of reagent/mol of average subunit when the ADP activation is completely lost and the enzyme is 68% inactivated. The time-dependent incorporation is consistent with the postulate that covalent reaction of 0.5 mol of BDB-TADP/mol of average enzyme subunit causes complete loss of ADP activation, while reaction with another 0.5 mol of BDB-TADP would lead to total inactivation. The enzyme is composed of three distinct subunits in the approximate ratio 2 alpha:1 beta:1 gamma. The distribution of BDB-[beta-32P]TADP incorporated into modified enzyme is 63:30:7% for alpha:beta:gamma throughout the course of the reaction. These results indicate the 6-(4-bromo-2,3-dioxobutyl)thioadenosine 5'-diphosphate functions as an affinity label of two types of potential metal-dependent ADP sites of NAD-dependent isocitrate dehydrogenase and that these allosteric sites are present on two (alpha and beta) of the enzyme's three types of subunits.
猪心NAD依赖型异柠檬酸脱氢酶受ADP变构激活，ADP可降低异柠檬酸的米氏常数。新型ADP类似物6-(4-溴-2,3-二氧代丁基)硫代腺苷5'-二磷酸(BDB-TADP)在pH 6.1和25℃下与该酶发生不可逆反应，导致在低异柠檬酸浓度下进行的测定中ADP增加初始速度的能力迅速丧失，而使用饱和异柠檬酸浓度时失活速度较慢。ADP激活丧失的速率常数对BDB-TADP浓度呈非线性依赖；在0.2 mM硫酸锰存在下，可逆酶-试剂复合物的抑制常数KI为0.069 mM，饱和试剂浓度下的最大反应速率常数kmax等于0.031 min-1。对于在导致整体失活的位点发生的反应，在0.2 mM硫酸锰存在下，初始可逆酶-试剂复合物的抑制常数KI估计为0.018 mM，最大反应速率常数kmax = 0.0083 min-1。1 mM ADP加0.2 mM硫酸锰或0.1 mM ADP加0.2 mM硫酸锰加0.2 mM异柠檬酸可完全保护酶免受两种反应的影响，但NAD、ATP或ADP加乙二胺四乙酸则不能。因此，BDB-TADP似乎修饰了两个不同的金属依赖性ADP结合位点。在0.2 mM硫酸锰存在下，于pH 6.1将异柠檬酸脱氢酶与0.14 mM BDB-[β-32P]TADP一起温育，当ADP激活完全丧失且酶失活68%时，平均每个亚基掺入0.81摩尔试剂。时间依赖性掺入与以下假设一致：平均每个酶亚基与0.5摩尔BDB-TADP发生共价反应会导致ADP激活完全丧失，而与另外0.5摩尔BDB-TADP反应会导致完全失活。该酶由三种不同的亚基组成，比例约为2α:1β:1γ。在整个反应过程中，掺入修饰酶中的BDB-[β-32P]TADP在α:β:γ亚基中的分布为63:30:7%。这些结果表明，6-(4-溴-2,3-二氧代丁基)硫代腺苷5'-二磷酸作为NAD依赖型异柠檬酸脱氢酶两种潜在金属依赖性ADP位点的亲和标记，并且这些别构位点存在于该酶三种亚基类型中的两种(α和β)上。
