Role of Lys-12 in Catalysis by Triosephosphate Isomerase: A Two-Part Substrate Approach
Role of Lys-12 in Catalysis by Triosephosphate Isomerase: A Two-Part Substrate Approach
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DOI：
10.1021/bi100538b
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发表时间：
2010-06-29
期刊：
影响因子：
2.9
通讯作者：
Richard, John P.
中科院分区：
文献类型：
--
作者：
Go, Maybelle K.;Koudelka, Astrid;Richard, John P.
我们报道了来自酿酒酵母的磷酸丙糖异构酶（TIM）中的K12 G突变导致（1）底物甘油醛3-磷酸（GAP）的Km增加了50倍，中间体类似物2-磷酸乙醇酸的竞争性抑制的Ki增加了60倍，这是由于Lys-12的烷基铵侧链和配体磷酸二价阴离子基团之间失去稳定基态相互作用所致;（2）GAP异构化的k（cat）降低了12000倍，表明从米氏络合物到过渡态，Lys-12侧链与底物之间的相互作用变紧;和（3）k（cat）/K-m降低6 × 10（5）倍，相当于Lys-12阳离子侧链对过渡态的总稳定性为7.8 kcal/mol。GAP在D2 O中的K12 G TIM催化异构化的四种产物的产率被定量为来自酶催化消除反应的磷酸二羟丙酮（DHAP）（27%）、[1（R）-H-2]DHAP（23%）、[2（R）-H-2]GAP（31%）和甲基乙二醛（18%）。K12 G突变对质子转移到D2 O中TIM结合的包合物中间体的三种产物的相对产率只有很小的影响，但它强烈地有利于催化消除反应得到甲基乙二醛。K12 G突变还导致结合乙醇醛（GA）异构化的k（cat）/K-m降低>= 14倍，尽管观察到的[1-C-13]GA在D2 O中的突变酶催化反应的主要产物是来自非特异性蛋白质催化反应的[1-C-13，2，2-di-H-2]GA。K12 G突变导致GAP和中性截短底物[1-C-13]GA反应的k（cat）/K-m大幅降低的观察结果为Lys-12的阳离子侧链和在糖底物“片段”去质子化的过渡态中在烯醇化物样氧处产生的负电荷之间的稳定相互作用提供了证据。
We report that the K12G mutation in triosephosphate isomerase (TIM) from Saccharomyces cerevisiae results in (1) a similar to 50-fold increase in K-m for the substrate glyceraldehyde 3-phosphate (GAP) and a 60-fold increase in K-i for competitive inhibition by the intermediate analogue 2-phosphoglycolate, resulting from the loss of stabilizing ground state interactions between the alkylammonium side chain of Lys-12 and the ligand phosphodianion group; (2) a 12000-fold decrease in k(cat) for isomerization of GAP, suggesting a tightening of interactions between the side chain of Lys-12 and the substrate on proceeding from the Michaelis complex to the transition state; and (3) a 6 x 10(5)-fold decrease in k(cat)/K-m, corresponding to a total 7.8 kcal/mol stabilization of the transition state by the cationic side chain of Lys-12. The yields of the four products of the K12G TIM-catalyzed isomerization of GAP in D2O were quantified as dihydroxyacetone phosphate (D H A P) (27%), [1(R)-H-2]DHAP (23%), [2(R)-H-2]GAP (31%), and methylglyoxal (18%) from an enzyme-catalyzed elimination reaction. The K12G mutation has only a small effect on the relative yields of the three products of the transfer of a proton to the TIM-bound enediol(ate) intermediate in D2O, but it strongly favors catalysis of the elimination reaction to give methylglyoxal. The K12G mutation also results in a >= 14-fold decrease in k(cat)/K-m for isomerization of bound glycolaldehyde (GA), although the dominant observed product of the mutant enzyme-catalyzed reaction of [1-C-13]GA in D2O is [1-C-13,2,2-di-H-2]GA from a nonspecific protein-catalyzed reaction. The observation that the K12G mutation results in a large decrease in k(cat)/K-m for the reactions of both GAP and the neutral truncated substrate [1-C-13]GA provides evidence for a stabilizing interaction between the cationic side chain of Lys-12 and the negative charge that develops at the enolate-like oxygen in the transition state for deprotonation of the sugar substrate "piece".