Enzyme architecture: the effect of replacement and deletion mutations of loop 6 on catalysis by triosephosphate isomerase.
Enzyme architecture: the effect of replacement and deletion mutations of loop 6 on catalysis by triosephosphate isomerase.
复制标题
DOI：
10.1021/bi500458t
复制
发表时间：
2014-06-03
期刊：
影响因子：
2.9
通讯作者：
Richard JP
中科院分区：
文献类型：
--
作者：
Zhai X;Go MK;O'Donoghue AC;Amyes TL;Pegan SD;Wang Y;Loria JP;Mesecar AD;Richard JP
检测了鸡肌磷酸丙糖异构酶（cTIM）中磷酸二阴离子夹环的两种突变：（1）通过去除残基170-173形成的环缺失突变体（LDM）[Pompliano，D. L.，等人（1990）Biochemistry 29，3186-3194]和（2）环6置换突变体（L 6 RM），其中来自真核生物的TIM的N-末端铰链序列166-PXW-168（X = L或V）被来自古细菌的序列166-PPE-168置换。L 6 RM的X射线晶体结构显示E168的侧链从野生型cTIM中的W168的侧链发生了大的位移。溶液核磁共振数据显示，L 6 RM导致6环及周围区域发生显着的化学位移变化，并且3-磷酸甘油（G3 P）的结合导致L 6 RM活性位点核的化学位移变化小于野生型cTIM的化学位移变化。与L 6 RM的环6的相互作用使烯二醇盐中间体朝向由LDM催化的消除反应稳定。LDM和L 6 RM导致GAP异构化的kcat/Km分别降低800000倍和23000倍。LDM的饱和，但不是L 6 RM，底物和抑制剂磷酸乙醇酸通过稳态动力学分析检测。我们建议，野生型TIM和L 6 RM的X-射线晶体结构的比较的基础上，配体结合弱的L 6 RM，因为大部分的配体结合能被用来克服不稳定的静电相互作用的侧链之间的E168和E129，预测开发的闭环酶。在GAP在D2 O中的LDM-和L 6 RM-催化反应中形成DHAP、d-DHAP和d-GAP的相似归一化产率。与野生型cTIM的这些产物的79%产率相比，在突变型cTIM催化的反应中观察到的较小的DHAP和d-DHAP的归一化12-13%产率表明这些突变损害了质子在初始烯二醇磷酸酯中间体处从O-2到O-1的转移。对于[1- 13 C]GA和HPi在D2 O中的LDM催化的异构化反应没有检测到产物，但是在0.020 M二价阴离子存在下的L 6 RM催化的反应得到2%的异构化产物[2- 13 C，2- 2 H]GA产率。
Two mutations of the phosphodianion gripper loop in chicken muscle triosephosphate isomerase (cTIM) were examined: (1) the loop deletion mutant (LDM) formed by removal of residues 170–173 [Pompliano, D. L., et al. (1990) Biochemistry 29, 3186–3194] and (2) the loop 6 replacement mutant (L6RM), in which the N-terminal hinge sequence of TIM from eukaryotes, 166-PXW-168 (X = L or V), is replaced by the sequence from archaea, 166-PPE-168. The X-ray crystal structure of the L6RM shows a large displacement of the side chain of E168 from that for W168 in wild-type cTIM. Solution nuclear magnetic resonance data show that the L6RM results in significant chemical shift changes in loop 6 and surrounding regions, and that the binding of glycerol 3-phosphate (G3P) results in chemical shift changes for nuclei at the active site of the L6RM that are smaller than those of wild-type cTIM. Interactions with loop 6 of the L6RM stabilize the enediolate intermediate toward the elimination reaction catalyzed by the LDM. The LDM and L6RM result in 800000- and 23000-fold decreases, respectively, in kcat/Km for isomerization of GAP. Saturation of the LDM, but not the L6RM, by substrate and inhibitor phosphoglycolate is detected by steady-state kinetic analyses. We propose, on the basis of a comparison of X-ray crystal structures for wild-type TIM and the L6RM, that ligands bind weakly to the L6RM because a large fraction of the ligand binding energy is utilized to overcome destabilizing electrostatic interactions between the side chains of E168 and E129 that are predicted to develop in the loop-closed enzyme. Similar normalized yields of DHAP, d-DHAP, and d-GAP are formed in LDM- and L6RM-catalyzed reactions of GAP in D2O. The smaller normalized 12–13% yield of DHAP and d-DHAP observed for the mutant cTIM-catalyzed reactions compared with the 79% yield of these products for wild-type cTIM suggests that these mutations impair the transfer of a proton from O-2 to O-1 at the initial enediolate phosphate intermediate. No products are detected for the LDM-catalyzed isomerization reactions in D2O of [1-13C]GA and HPi, but the L6RM-catalyzed reaction in the presence of 0.020 M dianion gives a 2% yield of the isomerization product [2-13C,2-2H]GA.
登录
查看更多内容
影响因子：
3.5
作者：
HERMES, JD;PAREKH, SM;KNOWLES, JR
通讯作者：
KNOWLES, JR
影响因子：
2.9
作者：
Go, Maybelle K.;Malabanan, M. Merced;Richard, John P.
通讯作者：
Richard, John P.
影响因子：
2.9
作者：
Go, Maybelle K.;Koudelka, Astrid;Richard, John P.
通讯作者：
Richard, John P.
影响因子：
2.7
作者：
DELAGLIO, F;GRZESIEK, S;BAX, A
通讯作者：
BAX, A
影响因子：
2.9
作者：
Grzesiek, S;Stahl, SJ;Bax, A
通讯作者：
Bax, A