Schliebs W, Thanki N, Eritja R, Wierenga R
European Molecular Biology Laboratory, Heidelberg, Germany.
Protein Sci. 1996 Feb;5(2):229-39. doi: 10.1002/pro.5560050206.
MonoTIM is a stable monomeric variant of the dimeric trypanosomal enzyme triose phosphate isomerase (TIM) with less, but significant, catalytic activity. It is known that in TIM, three residues, Lys 13 (loop 1), His 95 (loop 4), and Glu 167 (loop 6) are the crucial catalytic residues. In the wild-type TIM dimer, loop 1 and loop 4 are very rigid because of tight interactions with residues of the other subunit. Previous structural studies indicate that Lys 13 and His 95 have much increased conformational flexibility in monoTIM. Using site-directed mutagenesis, it is shown here that Lys 13 and His 95 are nevertheless essential for optimal catalysis by monoTIM: monoTIM-K13A is completely inactive, although it can still bind substrate analogues, and monoTIM-H95A is 50 times less active. The best inhibitors of wild-type TIM are phosphoglycolohydroxamate (PGH) and 2-phosphoglycolate (2PG), with KI values of 8 microM and 26 microM, respectively. The affinity of the monoTIM active site for PGH has been reduced approximately 60-fold, whereas for 2PG, only a twofold weakening of affinity is observed. The mode of binding, as determined by protein crystallographic analysis of these substrate analogues, shows that, in particular, 2PG interacts with Lys 13 and His 95 in a way similar but not identical to that observed for the wild-type enzyme. This crystallographic analysis also shows that Glu 167 has the same interactions with the substrate analogues as in the wild type. The data presented suggest that, despite the absence of the second subunit, monoTIM catalyzes the interconversion of D-glyceraldehyde-3-phosphate and dihydroxyacetone phosphate via the same mechanism as in the wild type.
单聚体三磷酸甘油醛异构酶（MonoTIM）是二聚体锥虫三磷酸甘油醛异构酶（TIM）的一种稳定单体变体，其催化活性较低但仍很显著。已知在TIM中，三个残基，即赖氨酸13（环1）、组氨酸95（环4）和谷氨酸167（环6）是关键的催化残基。在野生型TIM二聚体中，环1和环4由于与另一个亚基的残基紧密相互作用而非常刚性。先前的结构研究表明，在单聚体三磷酸甘油醛异构酶中赖氨酸13和组氨酸95的构象灵活性大大增加。本文通过定点诱变表明，赖氨酸13和组氨酸95对于单聚体三磷酸甘油醛异构酶的最佳催化作用仍然至关重要：单聚体三磷酸甘油醛异构酶-K13A完全无活性，尽管它仍然可以结合底物类似物，而单聚体三磷酸甘油醛异构酶-H95A的活性则低50倍。野生型TIM的最佳抑制剂是磷酸甘醇羟肟酸（PGH）和2-磷酸乙醇酸（2PG），其抑制常数（KI）值分别为8微摩尔/升和26微摩尔/升。单聚体三磷酸甘油醛异构酶活性位点对PGH的亲和力降低了约60倍，而对于2PG，仅观察到亲和力减弱两倍。通过对这些底物类似物的蛋白质晶体学分析确定的结合模式表明，特别是2PG与赖氨酸13和组氨酸95的相互作用方式与野生型酶相似但不完全相同。这种晶体学分析还表明，谷氨酸167与底物类似物的相互作用与野生型相同。所提供的数据表明，尽管没有第二个亚基，单聚体三磷酸甘油醛异构酶仍通过与野生型相同的机制催化3-磷酸-D-甘油醛和磷酸二羟丙酮的相互转化。
