Crystallography and site-directed mutagenesis of yeast triosephosphate isomerase: what can we learn about catalysis from a "simple" enzyme?
Crystallography and site-directed mutagenesis of yeast triosephosphate isomerase: what can we learn about catalysis from a "simple" enzyme?
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酵母磷酸三糖异构酶的晶体学和定点诱变：我们可以从“简单”酶的催化作用中学到什么？
DOI：
10.1101/sqb.1987.052.01.069
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发表时间：
1987
期刊：
影响因子：
--
通讯作者：
Ringe,D
中科院分区：
文献类型：
--
作者：
Alber,TC;DavenportJr,RC;Giammona,DA;Lolis,E;Petsko,GA;Ringe,D
如果有任何希望完全理解任何酶的催化作用，毫无疑问，最好的候选蛋白质是磷酸丙糖异构酶（TIM）。（按照诺尔斯和菲利普斯在牛津大学的研究小组最初建立的惯例，我们使用TIM作为酶的缩写，TPI代表其基因。）这种酶催化所有代谢生物化学中最简单的反应，即3-碳丙糖磷酸二羟丙酮磷酸（DHAP）和o-甘油醛-3-磷酸（o-GAP）的相互转化。该反应只是质子的转移，即DHAP的1号碳上的pro-R氢，立体专一地转移到2号碳上，形成GAP的邻位异构体（图1）。这两种糖磷酸的异构化是果糖1-6二磷酸的醛缩酶催化降解的产物，提供了ATP的净增益，使糖酵解成为有效的能量产生途径。由于只有o-GAP被糖酵解利用，TIM确保那些来自葡萄糖的碳原子最终在DHAP中被转化为GAP并向下汇集为丙酮酸，伴随着ATE的产生。该数值的大幅度是由于水合和未水合形式的D-GAP的表观Keq为22，水合平衡为29（Trentham等人，1969）;只有未水合形式的磷酸丙糖是异构酶的底物，甚至与异构酶结合（Webb等人，1977）。这种酶具有高度特异性。游离的丙糖不与酶结合;需要磷酸基团。二羟基丙酮硫酸盐不是底物，α-甘油磷酸盐是竞争性抑制剂（Wolfenden 1969）。
If there is any hope of completely understanding the catalytic action of any enzyme, surely the best candidate for the protein is triosephosphate isomerase (TIM).(Following a convention initially established by Knowles's and Phillips's groups at Oxford, we use TIM as an abbreviation for the enzyme and TPI to represent its gene.) This enzyme catalyzes the simplest reaction in all of metabolic biochemistry, the interconversion of the 3-carbon triosephosphates dihydroxyacetone phosphate (DHAP) and o-glyceraldehyde-3-phosphate (o-GAP). The reaction is just the transfer of a proton, the pro-R hydrogen from carbon 1 of DHAP, stereospecifically to carbon 2 to form the o-isomer of GAP (Fig. 1). Isomerization of these two sugar phosphates, which are the products of the aldolase-catalyzed degradation of fructose 1-6 biphosphate, provides the net gain of ATP that makes glycolysis an efficient energy-producing pathway. Since only o-GAP is utilized by glycolysis, TIM ensures that those carbon atoms from glucose that end up in DHAP are converted to GAP and funneled down to pyruvate, with concomitant production of ATEAlthough the equilibrium constant on the enzyme is not known, Keq for the overall reaction is 300 to 1 in favor of DHAP. The large magnitude of this number arises from the combination of an apparent Keq of 22 with a hydration equilibrium of 29 for the hydrated and unhydrated forms of D-GAP (Trentham et al. 1969); only the unhydrated forms of the triosephosphates are substrates for or even bind to the isomerase (Webb et al. 1977). The enzyme is highly specific. Free triose sugars do not bind to the enzyme; a phosphate group is required. Dihydroxyacetone sulfate is not a substrate, and a-glycerol phosphate is a competitive inhibitor (Wolfenden 1969).