Mechanism for Activation of Triosephosphate Isomerase by Phosphite Dianion: The Role of a Hydrophobic Clamp
Mechanism for Activation of Triosephosphate Isomerase by Phosphite Dianion: The Role of a Hydrophobic Clamp
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DOI：
10.1021/ja303695u
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发表时间：
2012-06-20
影响因子：
15
通讯作者：
Richard, John P.
中科院分区：
文献类型：
--
作者：
Malabanan, M. Merced;Koudelka, Astrid P.;Richard, John P.
研究了Ile-172和Leu-232疏水侧链在布鲁氏锥虫磷酸丙糖异构酶(TIM)催化3-磷酸甘油醛(GAP)可逆异构化合成磷酸二羟丙酮(DHAP)中的作用。I172a和L232a突变导致异构化反应的k(CAT)/K-m分别降低100倍和6倍。报道了突变对GAP和[1-C-13]-乙醇醛([1-C-13]-GA)在D2O中催化反应产物分布的影响。野生型TbB TIM催化的GAP异构化和分子内氢转移反应的DHAP产率分别为13%和4%，而I172A型和L232A型突变体的产率分别为13%和4%。同样，在I172a和L232a突变体催化的反应中，[1-C-13]-GA在D2O中异构化生成[2-C-13]-GA的产率分别降至2%和1%。氢分子内转移产物产率的下降与活性中心基团的重新定位一致，这有利于底物衍生的氢转移到结合中间体的蛋白质或氧阴离子。I172a和L232a突变导致(A)a>；对于TIM催化的[1-C-13]-GA在D2O中的反应，其二级速率常数降低了10倍(I172a)，增加了17倍(L232a)；(B)对于亚磷酸二阴离子(HPO32-)在D2O中TIM催化的反应，其三级速率常数降低了170倍(I172a)，增加了25倍(L232a)；I172a突变对整个底物和底物片段的野生TIM催化反应动力学参数的影响与突变酶的Glu-167羧酸侧链的碱性降低是一致的。这些数据提供了显著的证据表明，L232a突变导致催化活性的环闭合型TIM(E-C)相对于非活性开放型(E-O)的稳定化约1.7kcal/mol。
The role of the hydrophobic side chains of Ile-172 and Leu-232 in catalysis of the reversible isomerization of R-glyceraldehyde 3-phosphate (GAP) to dihydroxyacetone phosphate (DHAP) by triosephosphate isomerase (TIM) from Trypanosoma brucei brucei (Tbb) has been investigated. The I172A and L232A mutations result in 100- and 6-fold decreases in k(cat)/K-m for the isomerization reaction, respectively. The effect of the mutations on the product distributions for the catalyzed reactions of GAP and of [1-C-13]-glycolaldehyde ([1-C-13]-GA) in D2O is reported. The 40% yield of DHAP from wild-type Tbb TIM-catalyzed isomerization of GAP with intramolecular transfer of hydrogen is found to decrease to 13% and to 4%, respectively, for the reactions catalyzed by the I172A and L232A mutants. Likewise, the 13% yield of [2-C-13]-GA from isomerization of [1-C-13]-GA in D2O is found to decrease to 2% and to 1%, respectively, for the reactions catalyzed by the I172A and L232A mutants. The decrease in the yield of the product of intramolecular transfer of hydrogen is consistent with a repositioning of groups at the active site that favors transfer of the substrate-derived hydrogen to the protein or the oxygen anion of the bound intermediate. The I172A and L232A mutations result in (a) a >10-fold decrease (I172A) and a 17-fold increase (L232A) in the second-order rate constant for the TIM-catalyzed reaction of [1-C-13]-GA in D2O, (b) a 170-fold decrease (I172A) and 25-fold increase (L232A) in the third-order rate constant for phosphite dianion (HPO32-) activation of the TIM-catalyzed reaction of GA in D2O, and (c) a 1.5-fold decrease (I172A) and a larger 16-fold decrease (L232A) in K-d for activation of TIM by HPO32- in D2O. The effects of the I172A mutation on the kinetic parameters for the wild-type TIM-catalyzed reactions of the whole substrate and substrate pieces are consistent with a decrease in the basicity of the carboxylate side chain of Glu-167 for the mutant enzyme. The data provide striking evidence that the L232A mutation leads to a ca. 1.7 kcal/mol stabilization of a catalytically active loop-closed form of TIM (E-C) relative to an inactive open form (E-O).