Reiersen H, Sletten K, McKinley-McKee J S
Biochemical Institute, University of Oslo, Blindern, Norway.
Eur J Biochem. 1993 Feb 1;211(3):861-9. doi: 10.1111/j.1432-1033.1993.tb17619.x.
The metal-directed alkylating agent DL-alpha-bromo-beta-(5- imidazolyl)propionic acid (BrImPpOH) is shown to be an affinity-labelling reagent for sheep liver sorbitol dehydrogenase (SDH). As previously found for horse liver alcohol dehydrogenase (ADH), it modifies a cysteine ligand to the active-site zinc. In this case it is selectively incorporated (over 90%) at Cys43 in each of the four polypeptide chains/protomers of sheep liver SDH. Incorporated reagent and residual activity correlated. The first order inactivation constant, K2, and KEI, the dissociation constant for SDH and BrImPpOH, have been determined at different pH. The reactivity of BrImPpOH for SDH is higher than that for horse liver and yeast ADH. The protection of SDH against BrImPpOH inactivation by buffers and other molecules shows some similarities to that with horse liver ADH. However, sheep liver SDH bound BrImPpOH, imidazole and phosphate ions much weaker than liver ADH. The pKa values from the plot of log (k2/KEI) against pH are approximately 7.0 and 8.8-8.9. The former pKa value probably represents ionization of an imidazole group and the latter the zinc/water ionization in SDH. These pKa values are similar to those found for horse liver ADH. They are apparently not noticeably influenced by a second cysteine ligand in liver ADH being replaced by a proposed glutamic acid residue as a ligand to the catalytic zinc in SDH. The plot of logk2 against pH shows pKa values around 7.0 and 9.2 for the SDH-BrImPpOH-complex. The pKa of 7.0 is the same as for log(k2/KEI), and indicates no significant perturbation due to the binding of BrImPpOH to SDH. The pKa around 9.2 indicates perturbation of the zinc/water ionization or the ionization of Cys43.
金属导向烷基化剂DL-α-溴-β-(5-咪唑基)丙酸(BrImPpOH)被证明是绵羊肝脏山梨醇脱氢酶(SDH)的一种亲和标记试剂。正如之前在马肝脏乙醇脱氢酶(ADH)中发现的那样，它修饰了活性位点锌的半胱氨酸配体。在这种情况下，它在绵羊肝脏SDH的四条多肽链/原体中的每一条链的Cys43处被选择性掺入(超过90%)。掺入的试剂与残余活性相关。已在不同pH值下测定了一级失活常数K2以及SDH与BrImPpOH的解离常数KEI。BrImPpOH对SDH的反应性高于对马肝脏和酵母ADH的反应性。缓冲液和其他分子对SDH免受BrImPpOH失活的保护作用与对马肝脏ADH的保护作用有一些相似之处。然而，绵羊肝脏SDH结合BrImPpOH、咪唑和磷酸根离子的能力比肝脏ADH弱得多。log(k2/KEI)对pH的曲线图中的pKa值约为7.0和8.8 - 8.9。前一个pKa值可能代表咪唑基团的电离，后一个代表SDH中锌/水的电离。这些pKa值与在马肝脏ADH中发现的相似。显然，肝脏ADH中第二个半胱氨酸配体被提议作为SDH中催化锌配体的谷氨酸残基取代后，它们没有受到明显影响。logk2对pH的曲线图显示SDH - BrImPpOH复合物的pKa值在7.0和9.2左右。7.0的pKa与log(k2/KEI)的相同，表明BrImPpOH与SDH结合没有显著扰动。9.2左右的pKa表明锌/水的电离或Cys43的电离受到了扰动。
