Karsten William E, Pais June E, Rao G S Jagannatha, Harris Ben G, Cook Paul F
Department of Chemistry and Biochemistry, University of Oklahoma, 620 Parrington Oval, Norman, Oklahoma 73019, USA.
Biochemistry. 2003 Aug 19;42(32):9712-21. doi: 10.1021/bi034101w.
The kinetic mechanism of activation of the mitochondrial NAD-malic enzyme from the parasitic roundworm Ascaris suum has been studied using a steady-state kinetic approach. The following conclusions are suggested. First, malate and fumarate increase the activity of the enzyme in both reaction directions as a result of binding to separate allosteric sites, i.e., sites that exist in addition to the active site. The binding of malate and fumarate is synergistic with the K(act) decreasing by >or=10-fold at saturating concentrations of the other activator. Second, the presence of the activators decreases the K(m) for pyruvate 3-4-fold, and the K(i) (Mn) >or=20-fold in the direction of reductive carboxylation; similar effects are obtained with fumarate in the direction of oxidative decarboxylation. The greatest effect of the activators is thus expressed at low reactant concentrations, i.e., physiologic concentrations of reactant, where activation of >or=15-fold is observed. A recent crystallographic structure of the human mitochondrial NAD malic enzyme [13] shows fumarate bound to an allosteric site. Site-directed mutagenesis was used to change R105, homologous to R91 in the fumarate activator site of the human enzyme, to alanine. The R105A mutant enzyme exhibits the same maximum rate and V/K(NAD) as does the wild-type enzyme, but 7-8-fold decrease in both V/K(malate) and V/K(Mg), indicating the importance of this residue in the activator site. In addition, neither fumarate nor malate activates the enzyme in either reaction direction. Finally, a change in K143 (a residue in a positive pocket adjacent to that which contains R105), to alanine results in an increase in the K(act) for malate by about an order of magnitude such that it is now of the same magnitude as the K(m) for malate. The K143A mutant enzyme also exhibits an increase in the K(act) for fumarate (in the absence of malate) from 200 microM to about 25 mM.
采用稳态动力学方法研究了寄生蛔虫猪蛔虫线粒体NAD - 苹果酸酶的激活动力学机制。得出以下结论。首先，苹果酸和富马酸通过与不同的变构位点结合，在两个反应方向上均增加了该酶的活性，即除活性位点外存在的位点。苹果酸和富马酸的结合具有协同作用，在另一种激活剂饱和浓度下，K(act)降低≥10倍。其次，激活剂的存在使丙酮酸的K(m)降低3 - 4倍，在还原羧化方向上K(i)(Mn)降低≥20倍；在氧化脱羧方向上富马酸也有类似作用。因此，激活剂的最大作用在低反应物浓度时表现出来，即反应物的生理浓度时，观察到激活倍数≥15倍。人线粒体NAD苹果酸酶的最新晶体结构[13]显示富马酸结合在一个变构位点上。定点诱变用于将与人类酶富马酸激活位点中的R91同源的R105突变为丙氨酸。R105A突变酶与野生型酶具有相同的最大反应速率和V/K(NAD)，但V/K(苹果酸)和V/K(Mg)均降低7 - 8倍，表明该残基在激活位点中的重要性。此外，富马酸和苹果酸在任一反应方向上均不激活该酶。最后，将K143（与包含R105的口袋相邻的正口袋中的一个残基）突变为丙氨酸，导致苹果酸的K(act)增加约一个数量级，使其现在与苹果酸的K(m)大小相同。K143A突变酶在（无苹果酸时）富马酸的K(act)也从200μM增加到约25 mM。
