Susan-Resiga Delia, Nowak Thomas
Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, USA.
Biochemistry. 2004 Dec 7;43(48):15230-45. doi: 10.1021/bi049864d.
The active site T298 residue of yeast pyruvate kinase (YPK), located in a position to serve potentially as the proton donor, was mutated to cysteine. T298C YPK was isolated and purified, and its enzymatic properties were characterized. Fluorescence and CD spectra indicate minor structural perturbations. A kinetic analysis of the Mg(2+)-activated enzyme demonstrates no catalytic activity in the absence of the heterotropic activator fructose 1,6-bisphosphate (FBP). In the presence of Mg(2+) and FBP, T298C has approximately 20% of the activity of wild-type (wt) YPK. The activator constant for FBP increases by 1 order of magnitude compared to this constant with the wt enzyme. T298C shows positive cooperativity by FBP with a Hill coefficient of 2.6 (wt, n(H,FBP) = 1). Mn(2+)-activated T298C behaves like Mn(2+)-activated wt YPK with a V(max) that is 20% of that for the wt enzyme with or without FBP. A pH-rate profile of T298C relative to that for wt YPK shows that pK(a,2) has shifted from 6.4 in wt to 5.5, indicating that the thiol group elicits an acidic pK shift. Inactivation of both wt and T298C by iodoacetate elicits a pseudo-first-order loss of activity with T298C being inactivated from 8 to 100 times faster than wt YPK. A pH dependence of the inactivation rate constant for T298C gives a value of 8.2, consistent with the pK for a thiol. Changes in fluorescence indicate that the T298C-Mg(2+) complex binds PEP, ADP, and both ligands together. This demonstrates that the lack of activity is not due to the loss of substrate binding but to the lack of ability to induce the proper conformational change. The mutation also induces changes in binding of FBP to all the relevant complexes. Binding of the metal and binding of PEP to the enzyme complexes are also differentially altered. Solvent isotope effects are observed for both wt and T298C. Proton inventory studies indicate that k(cat) is affected by a proton from water in the transition state and the effects are metal ion-dependent. The results are consistent with water being the active site proton donor. Active site residue T298 is not critical for activity but plays a role in the activation of the water and affects the pK that modulates catalytic activity.
酵母丙酮酸激酶（YPK）的活性位点T298残基位于可能作为质子供体的位置，被突变为半胱氨酸。分离并纯化了T298C YPK，并对其酶学性质进行了表征。荧光和圆二色光谱表明结构扰动较小。对Mg(2+)激活的酶进行动力学分析表明，在没有异源激活剂果糖1,6-二磷酸（FBP）的情况下没有催化活性。在存在Mg(2+)和FBP的情况下，T298C的活性约为野生型（wt）YPK的20%。与wt酶相比，FBP的激活常数增加了1个数量级。T298C显示出FBP的正协同性，希尔系数为2.6（wt，n(H,FBP)=1）。Mn(2+)激活的T298C的行为类似于Mn(2+)激活的wt YPK，其V(max)是有或没有FBP时wt酶的20%。T298C相对于wt YPK的pH速率曲线表明，pK(a,2)已从wt中的6.4移至5.5，表明硫醇基团引起酸性pK位移。碘乙酸对wt和T298C的失活引发了活性的假一级损失，T298C的失活速度比wt YPK快8至100倍。T298C失活速率常数的pH依赖性给出的值为8.2，与硫醇的pK一致。荧光变化表明T298C-Mg(2+)复合物结合磷酸烯醇式丙酮酸（PEP）、二磷酸腺苷（ADP）以及两种配体。这表明缺乏活性不是由于底物结合的丧失，而是由于缺乏诱导适当构象变化的能力。该突变还诱导了FBP与所有相关复合物结合的变化。金属的结合以及PEP与酶复合物的结合也有差异地改变。wt和T298C均观察到溶剂同位素效应。质子存量研究表明，k(cat)在过渡态受到来自水的质子的影响，且这些影响是金属离子依赖性的。结果与水是活性位点质子供体一致。活性位点残基T298对活性不是至关重要的，但在水的激活中起作用，并影响调节催化活性的pK。
