Diefenbach R J, Candy J M, Mattick J S, Duggleby R G
Department of Biochemistry, University of Queensland, Brisbane, Australia.
FEBS Lett. 1992 Jan 13;296(1):95-8. doi: 10.1016/0014-5793(92)80411-9.
A tryptophan residue at position 487 in Zymomonas mobilis pyruvate decarboxylase was altered to leucine by site-directed mutagenesis. This modified Z. mobilis pyruvate decarboxylase was active when expressed in Escherichia coli and had unchanged kinetics towards pyruvate. The enzyme showed a decreased affinity for the cofactors with the half-saturating concentrations increasing from 0.64 to 9.0 microM for thiamin diphosphate and from 4.21 to 45 microM for Mg2+. Unlike the wild-type enzyme, there was little quenching of tryptophan fluorescence upon adding cofactors to this modified form. The data suggest that tryptophan-487 is close to the cofactor binding site but is not required absolutely for pyruvate decarboxylase activity. Substitution of asparagine, threonine or glycine for aspartate-440, a residue which is conserved between many thiamin diphosphate-dependent enzymes, completely abolishes enzyme activity.
通过定点诱变将运动发酵单胞菌丙酮酸脱羧酶中第487位的色氨酸残基替换为亮氨酸。这种修饰后的运动发酵单胞菌丙酮酸脱羧酶在大肠杆菌中表达时具有活性，并且对丙酮酸的动力学特性未发生改变。该酶对辅因子的亲和力降低，硫胺素二磷酸的半饱和浓度从0.64微摩尔增加到9.0微摩尔，镁离子的半饱和浓度从4.21微摩尔增加到45微摩尔。与野生型酶不同，向这种修饰形式中添加辅因子时，色氨酸荧光几乎没有淬灭。数据表明，色氨酸-487靠近辅因子结合位点，但对于丙酮酸脱羧酶活性并非绝对必需。将许多依赖硫胺素二磷酸的酶之间保守的天冬氨酸-440残基替换为天冬酰胺、苏氨酸或甘氨酸，会完全消除酶活性。
