Laboratoire de Chimie Bioorganique et Bioinorganique, ICMMO, Univ Paris-Sud, UMR 8182, Orsay F-91405, France.
Proteins. 2011 Jan;79(1):203-20. doi: 10.1002/prot.22873.
Type I phosphomannose isomerases (PMIs) are zinc-dependent metalloenzymes involved in the reversible isomerization of D-mannose 6-phosphate (M6P) and D-fructose 6-phosphate (F6P). 5-Phospho-D-arabinonohydroxamic acid (5PAH), an inhibitor endowed with nanomolar affinity for yeast (Type I) and Pseudomonas aeruginosa (Type II) PMIs (Roux et al., Biochemistry 2004; 43:2926-2934), strongly inhibits human (Type I) PMI (for which we report an improved expression and purification procedure), as well as Escherichia coli (Type I) PMI. Its K(i) value of 41 nM for human PMI is the lowest value ever reported for an inhibitor of PMI. 5-Phospho-D-arabinonhydrazide, a neutral analogue of the reaction intermediate 1,2-cis-enediol, is about 15 times less efficient at inhibiting both enzymes, in accord with the anionic nature of the postulated high-energy reaction intermediate. Using the polarizable molecular mechanics, sum of interactions between fragments ab initio computed (SIBFA) procedure, computed structures of the complexes between Candida albicans (Type I) PMI and the cyclic substrate β-D-mannopyranose 6-phosphate (β-M6P) and between the enzyme and the high-energy intermediate analogue inhibitor 5PAH are reported. Their analysis allows us to identify clearly the nature of each individual active site amino acid and to formulate a hypothesis for the overall mechanism of the reaction catalyzed by Type I PMIs, that is, the ring-opening and isomerization steps, respectively. Following enzyme-catalyzed ring-opening of β-M6P by zinc-coordinated water and Gln111 ligands, Lys136 is identified as the probable catalytic base involved in proton transfer between the two carbon atoms C1 and C2 of the substrate D-mannose 6-phosphate.
I 型磷酸甘露糖异构酶（PMIs）是一种锌依赖性金属酶，参与 D-甘露糖 6-磷酸（M6P）和 D-果糖 6-磷酸（F6P）的可逆异构化。5-磷酸-D-阿拉伯庚酮糖羟肟酸（5PAH）是一种对酵母（I 型）和铜绿假单胞菌（II 型）PMIs 具有纳摩尔亲和力的抑制剂（Roux 等人，生物化学 2004 年；43:2926-2934），强烈抑制人（I 型）PMI（我们报告了一种改进的表达和纯化程序）以及大肠杆菌（I 型）PMI。它对人 PMI 的 K(i)值为 41 nM，是 PMI 抑制剂的最低值。5-磷酸-D-阿拉伯庚酮酰肼，反应中间体 1,2-顺-enediol 的中性类似物，对两种酶的抑制效率约低 15 倍，这与假定的高能反应中间体的阴离子性质一致。使用极化分子力学，片段间相互作用总和从头计算（SIBFA）程序，报道了白色念珠菌（I 型）PMI 与环状底物β-D-甘露吡喃糖 6-磷酸（β-M6P）和酶与高能中间体类似物抑制剂 5PAH 之间的复合物的计算结构。它们的分析使我们能够清楚地识别每个活性位点氨基酸的性质，并提出 I 型 PMIs 催化反应的总体机制假说，即分别为开环和异构化步骤。在锌配位水和 Gln111 配体催化β-M6P 开环之后，鉴定 Lys136 为参与底物 D-甘露糖 6-磷酸的两个碳原子 C1 和 C2 之间质子转移的可能催化碱。
