Misset O, Opperdoes F R
Eur J Biochem. 1987 Feb 2;162(3):493-500. doi: 10.1111/j.1432-1033.1987.tb10667.x.
Trypanosoma brucei has two phosphoglycerate kinase (PGK) isoenzymes, one is particle-bound and localized in glycosomes while the other is present in the cytosol. The cytosolic isoenzyme (cPGK) was 900-fold purified from cultured procyclic trypanosomes by hydrophobic interaction chromatography on phenyl-Sepharose followed by affinity chromatography on 2',3'-ATP-Sepharose and had a specific activity of 275 units/mg protein. cPGK was compared with the purified glycosomal isoenzyme (gPGK) from bloodstream-form trypanosomes as well as with the commercially available PGKs from yeast, rabbit muscle and Spirulina platensis, a blue-green alga. Like all other PGKs, cPGK was a monomeric protein with a molecular mass of approximately 45 kDa similar to that of the PGKs from other organisms but 2 kDa smaller than that of gPGK. Despite this difference in length and a great difference in isoelectric point, the two trypanosome isoenzymes strongly resembled each other in several respects. The kinetic parameters did not differ significantly from each other or from the PGKs of other organisms. Both trypanosome enzymes resembled the enzyme from S. platensis in that they had an almost absolute requirement for ATP, contrary to the enzymes from yeast and rabbit muscle, which were capable of utilizing GTP and ITP also. This difference in substrate specificity may be related to the amino acid substitutions, Trp 308----His and Ala 306----Glu in the adenine-binding site, which are only found in the two Trypanosoma isoenzymes. Kinetic analysis showed that these substitutions do not prevent binding of the ATP analogues, but probably prevent phosphoryl-group transfer. Both isoenzymes displayed an activity optimum at pH 6.0-9.0 similar to that for the enzyme of yeast. Both gPGK and cPGK were inhibited by the trypanocidal drug Suramin. This inhibition could be described as competitive both with ATP and 3-phosphoglycerate with two inhibitor molecules binding to one molecule of enzyme. The gPGK, however, was much more sensitive (Ki app. = 8.0 microM) to Suramin than either the cPGK (Ki app. = 20 microM) or the enzymes from rabbit muscle (Ki app. = 55 microM), yeast (Ki app. = 167 microM) or S. platensis (Ki app. = 250 microM). It is suggested that positive charges on the enzyme's surface may play an important role in the potentiation of the binding of the negatively charged Suramin molecule.
布氏锥虫有两种磷酸甘油酸激酶（PGK）同工酶，一种与颗粒结合并定位于糖体中，另一种存在于胞质溶胶中。通过在苯基琼脂糖上进行疏水相互作用色谱，随后在2'，3'-ATP-琼脂糖上进行亲和色谱，从培养的前循环锥虫中纯化出胞质同工酶（cPGK）900倍，其比活性为275单位/毫克蛋白质。将cPGK与来自血流型锥虫的纯化糖体同工酶（gPGK）以及来自酵母、兔肌肉和蓝藻钝顶螺旋藻的市售PGK进行了比较。与所有其他PGK一样，cPGK是一种单体蛋白，分子量约为45 kDa，与其他生物的PGK相似，但比gPGK小2 kDa。尽管在长度上存在这种差异且等电点差异很大，但两种锥虫同工酶在几个方面彼此非常相似。动力学参数彼此之间以及与其他生物的PGK相比没有显著差异。两种锥虫酶与钝顶螺旋藻的酶相似，即它们几乎绝对需要ATP，这与酵母和兔肌肉的酶相反，后者也能够利用GTP和ITP。这种底物特异性的差异可能与腺嘌呤结合位点中的氨基酸取代Trp 308→His和Ala 306→Glu有关，这仅在两种锥虫同工酶中发现。动力学分析表明，这些取代并不妨碍ATP类似物的结合，但可能妨碍磷酸基团转移。两种同工酶在pH 6.0 - 9.0时均显示出与酵母酶相似的活性最佳值。gPGK和cPGK均被杀锥虫药物苏拉明抑制。这种抑制可被描述为与ATP和3-磷酸甘油酸均具有竞争性，两个抑制剂分子与一个酶分子结合。然而，gPGK对苏拉明比cPGK（Ki app. = 20 microM）或兔肌肉（Ki app. = 55 microM）、酵母（Ki app. = 167 microM）或钝顶螺旋藻（Ki app. = 250 microM）的酶更敏感（Ki app. = 8.0 microM）。有人提出，酶表面的正电荷可能在带负电荷的苏拉明分子结合的增强中起重要作用。
