pH variation of the kinetic parameters and the catalytic mechanism of malic enzyme.
pH variation of the kinetic parameters and the catalytic mechanism of malic enzyme.
复制标题
苹果酸酶动力学参数的pH变化及其催化机制。
作者：
M. Schimerlik;W. Cleland
利用pH值对苹果酸酶催化L-苹果酸氧化脱羧和乙酸乙酯脱羧反应动力学参数的影响，探讨了苹果酸酶的催化机理。与Mn 2+作为激活剂，具有pK为5.4的活性位点残基必须被质子化以用于乙酸乙酯脱羧和离子化以用于L-苹果酸的氧化脱羧。以Mg 2+为金属，该pK为6，并且在高pH下，当pK为7.8和9的基团被去质子化时，L-苹果酸的V/K降低。7.8处的基团是中性酸（被认为是与Mg 2+配位的水），而9处的基团是阳离子酸，如赖氨酸。苹果酸盐反应的V曲线显示这些pKs向外位移1.4个pH单位，因为限速步骤通常是TPNH释放，并且pH敏感的化学反应快25倍。通过pK 9.3基团的电离或pK 5.3基团的质子化降低TPN结合。的Km为镁的pH值的变化表明，质子化的一个组与pK 8.7（可能SH）减少金属结合在苹果酸盐的存在下，由一个因素的1400，并在没有苹果酸的情况下，由一个因素的20。提出了一种催化机制，其中氢化物转移伴随着质子转移到pK为5.4-6的基团，并且烯醇丙酮酸在脱羧和释放CO2后被配位到Mg 2+（pK 7.8）的水质子化。
The pH variation of the kinetic parameters for the oxidative decarboxylation of L-malate and decarboxylation of oxalacetate catalyzed by malic enzyme has been used to gain information on the catalytic mechanism of this enzyme. With Mn2+ as the activator, an active-site residue with a pK of 5.4 must be protonated for oxalacetate decarboxylation and ionized for the oxidative decarboxylation of L-malate. With Mg2+ as the metal, this pK is 6, and, at high pH, V/K for L-malate decreases when groups with pKs of 7.8 and 9 are deprotonated. The group at 7.8 is a neutral acid (thought to be water coordinated to Mg2+), while the group at 9 is a cationic acid such as lysine. The V profile for reaction of malate shows these pKs displaced outward by 1.4 pH units, since the rate-limiting step is normally TPNH release, and the chemical reaction, which is pH sensitive, is 25 times faster. TPN binding is decreased by ionization of a group with pK 9.3 or protonation of a group with pK 5.3. The pH variation of the Km for Mg shows that protonation of a group with pK 8.7 (possibly SH) decreases metal binding in the presence of malate by a factor of 1400, and in the absence of malate by a factor of 20. A catalytic mechanism is proposed in which hydride transfer is accompanied by transfer of a proton to the group with pK 5.4-6, and enolpyruvate is protonated by water coordinated to the Mg2+ (pK 7.8) after decarboxylation and release of CO2.