Lambeir A M, Loiseau A M, Kuntz D A, Vellieux F M, Michels P A, Opperdoes F R
International Institute of Cellular and Molecular Pathology, Research Unit for Tropical Diseases, Brussels, Belgium.
Eur J Biochem. 1991 Jun 1;198(2):429-35. doi: 10.1111/j.1432-1033.1991.tb16032.x.
The protozoan haemoflagellate Trypanosoma brucei has two NAD-dependent glyceraldehyde-3-phosphate dehydrogenase isoenzymes, each with a different localization within the cell. One isoenzyme is found in the cytosol, as in other eukaryotes, while the other is found in the glycosome, a microbody-like organelle that fulfils an essential role in glycolysis. The kinetic properties of the purified glycosomal and cytosolic isoenzymes were compared with homologous enzymes from other organisms. Both trypanosome enzymes had pH/activity profiles similar to that of other glyceraldehyde-3-phosphate dehydrogenases, with optimal activity around pH 8.5-9. Only the yeast enzyme showed its maximal activity at a lower pH. The glycosomal enzyme was more sensitive to changes in ionic strength below 0.1 M, while the cytosolic enzyme resembled more the enzymes from rabbit muscle, human erythrocytes and yeast. The affinity for NAD of the glycosomal enzyme was 5-10-fold lower than that of the cytosolic, as well as the other enzymes. A similar, but less pronounced, difference was found for its affinity for NADH. These differences are explained by a number of amino acid substitutions in the NAD-binding domain of the glycosomal isoenzyme. In addition, the effects of suramin, gossypol, agaricic acid and pentalenolactone on the trypanosome enzymes were studied. The trypanocidal drug suramin inhibited both enzymes, but in a different manner. Inhibition of the cytosolic enzyme was competitive with NAD, while in the case of the glycosomal isoenzyme, with NAD as substrate, the drug had an effect both on Km and Vmax. The most potent inhibitor was pentalenolactone, which at micromolar concentrations inhibited the glycosomal enzyme and the enzymes from yeast and Bacillus stearothermophilus in a reversible manner, while the rabbit muscle enzyme was irreversibly inhibited.
原生动物血鞭毛虫布氏锥虫有两种依赖NAD的3-磷酸甘油醛脱氢酶同工酶，每种在细胞内的定位都不同。一种同工酶存在于胞质溶胶中，如同在其他真核生物中一样，而另一种存在于糖体中，糖体是一种类似微体的细胞器，在糖酵解中起关键作用。将纯化的糖体和胞质溶胶同工酶的动力学特性与来自其他生物体的同源酶进行了比较。两种锥虫酶的pH/活性曲线与其他3-磷酸甘油醛脱氢酶相似，在pH 8.5 - 9左右具有最佳活性。只有酵母酶在较低pH下显示出最大活性。糖体酶在离子强度低于0.1 M时对离子强度变化更敏感，而胞质溶胶酶更类似于来自兔肌肉、人红细胞和酵母的酶。糖体酶对NAD的亲和力比胞质溶胶酶以及其他酶低5 - 10倍。在其对NADH的亲和力方面也发现了类似但不太明显的差异。这些差异是由糖体同工酶的NAD结合结构域中的一些氨基酸取代所解释的。此外，还研究了苏拉明、棉酚、木耳酸和戊烯内酯对锥虫酶的影响。杀锥虫药物苏拉明抑制这两种酶，但方式不同。对胞质溶胶酶的抑制与NAD竞争，而对于糖体同工酶，以NAD为底物时，该药物对Km和Vmax都有影响。最有效的抑制剂是戊烯内酯，它在微摩尔浓度下以可逆方式抑制糖体酶以及来自酵母和嗜热脂肪芽孢杆菌的酶，而兔肌肉酶则被不可逆地抑制。
