Kinetic characterization of yeast pyruvate carboxylase isozyme pyc1 and the pyc1 mutant, C249A
Kinetic characterization of yeast pyruvate carboxylase isozyme pyc1 and the pyc1 mutant, C249A
复制标题
DOI：
10.1021/bi035575y
复制
发表时间：
2004-02-03
期刊：
影响因子：
2.9
通讯作者：
Attwood, PV
中科院分区：
文献类型：
--
作者：
Branson, JP;Nezic, M;Attwood, PV
丙酮酸羧化酶的酵母Pyc 1同种型已被进一步表征，并显示出与Pyc 2同种型在其K+活化的K-a上不同。Pyc 1与鸡肝丙酮酸羧化酶的不同之处在于乙酰辅酶A对氨基甲酰磷酸ADP磷酸化缺乏影响，这可能是两种酶之间效应物作用位点差异的结果。溶剂D2 O同位素的影响已被测量与Pyc 1上的完整的丙酮酸羧化反应，在丙酮酸的情况下的ATP酶反应，和第一次为丙酮酸羧化酶的氨甲酰磷酸-ADP磷酸化反应。质子库存表明，所测量的同位素效应是由于在反应中的一个单一的质子转移步骤。在所有反应中观察到的逆同位素效应表明质子转移步骤将酶从非活性形式转化为活性形式。对C249 A突变酶的动力学测量表明，C249参与酶激活剂K+和乙酰辅酶A的结合和作用。C249不参与ATP结合，如在大肠杆菌乙酰辅酶A羧化酶的生物素羧化酶亚基中观察到的相应残基，也不直接负责测量的逆（D）（k（cat）/K-m）同位素效应。反同位素效应的大小表明，它们可能是由于形成低势垒氢键。用邻苯二甲醛修饰野生型和C249 A突变体表明，C249参与异吲哚的形成，但该残基的修饰并不直接导致伴随的酶活性的主要损失。
The yeast Pyc1 isoform of pyruvate carboxylase has been further characterized and shown to differ from the Pyc2 isoform in its K-a for K+ activation. Pyc1 differs from chicken liver pyruvate carboxylase in the lack of effect of acetyl-CoA on ADP phosphorylation by carbamoyl phosphate, which may be a result of differences in the loci of action of the effector between the two enzymes. Solvent D2O isotope effects have been measured with Pyc1 on the full pyruvate carboxylation reaction, the ATPase reaction in the absence of pyruvate, and the carbamoyl phosphate-ADP phosphorylation reaction for the first time for pyruvate carboxylase. Proton inventories indicate that the measured isotope effects are due to a single proton transfer step in the reaction. The inverse isotope effects observed in all reactions suggest that the proton transfer step converts the enzyme from an inactive to an active form. Kinetic measurements on the C249A mutant enzyme suggest that C249 is involved in the binding and action of enzyme activators K+ and acetyl-CoA. C249 is not involved in ATP binding as was observed for the corresponding residue in the biotin carboxylase subunit of Escherichia coli acetyl-CoA carboxylase, nor is it directly responsible for the measured inverse (D)(k(cat)/K-m) isotope effects. The size of the inverse isotope effects indicates that they may result from formation of a low-barrier hydrogen bond. Modification of the wild type and C249A mutant with o-phthalaldehyde suggests that C249 is involved in isoindole formation but that the modification of this residue is not directly responsible for the accompanying major loss of enzyme activity.