Candy J M, Koga J, Nixon P F, Duggleby R G
Department of Biochemistry, University of Queensland, Brisbane, Australia.
Biochem J. 1996 May 1;315 ( Pt 3)(Pt 3):745-51. doi: 10.1042/bj3150745.
Several enzymes require thiamine diphosphate (ThDP) as an essential cofactor, and we have used one of these, pyruvate decarboxylase (PDC; EC 4.1.1.1) from Zymomonas mobilis, as a model for this group of enzymes. It is well suited for this purpose because of its stability, ease of purification, homotetrameric subunit structure and simple kinetic properties. Crystallographic analyses of three ThDP-dependent enzymes [Müller, Lindqvist, Furey, Schulz, Jordan and Schneider (1993) Structure 1, 95-103] have suggested that an invariant glutamate participates in catalysis. In order to evaluate the role of this residue, identified in PDC from Zymomonas mobilis as Glu-50, it has been altered to glutamine and aspartate by site-directed mutagenesis of the cloned gene. The mutant proteins were expressed in Escherichia coli. Here we demonstrate that substitution with aspartate yields an enzyme with 3% of the activity of the wild-type, but with normal kinetics for pyruvate. Replacement of Glu-50 with glutamine yields an enzyme with only 0.5% of the catalytic activity of the wild-type enzyme. Each of these mutant enzymes has a decreased affinity for both ThDP and Mg2+. It has been reported that the binding of cofactors to apoPDC quenches the intrinsic tryptophan fluorescence [Diefenbach and Duggleby (1991) Biochem. J. 276, 439-445] and we have identified the residue responsible as Trp-487 [Diefenbach, Candy, Mattick and Duggleby (1992) FEBS Lett. 296, 95-98]. Although this residue is some distance from the cofactor binding site, it lies in the dimer interface, and the proposal has been put forward [Dyda, Furey, Swaminathan, Sax, Farrenkopf and Jordan (1993) Biochemistry 32, 6165-6170] that alteration of ring stacking with Phe-496 of the adjacent subunit is the mechanism of fluorescence quenching when cofactors bind. The closely related enzyme indolepyruvate decarboxylase (from Enterobacter cloacae) has a leucine residue at the position corresponding to Phe-496 but shows fluorescence quenching properties that are similar to those of PDC. This suggests that the fluorescence quenching is due to some perturbation of the local environment of Trp-487 rather than to a specific interaction with Phe-496. This latter hypothesis is supported by our data: mutation of this phenylalanine to leucine, isoleucine or histidine in PDC does not eliminate the fluorescence quenching upon addition of cofactors.
几种酶需要硫胺素二磷酸（ThDP）作为必需的辅因子，我们选用了其中一种酶——来自运动发酵单胞菌的丙酮酸脱羧酶（PDC；EC 4.1.1.1）作为这类酶的模型。由于其稳定性、易于纯化、同四聚体亚基结构和简单的动力学特性，它非常适合用于此目的。对三种依赖ThDP的酶进行的晶体学分析[Müller、Lindqvist、Furey、Schulz、Jordan和Schneider（1993年）《结构》1，95 - 103]表明，一个不变的谷氨酸参与催化作用。为了评估在运动发酵单胞菌的PDC中被鉴定为Glu - 50的这个残基的作用，通过对克隆基因进行定点诱变，将其改变为谷氨酰胺和天冬氨酸。突变蛋白在大肠杆菌中表达。在此我们证明，用天冬氨酸替代产生的一种酶具有野生型活性的3%，但对丙酮酸具有正常的动力学特性。用谷氨酰胺替代Glu - 50产生的一种酶仅具有野生型酶催化活性的0.5%。这些突变酶中的每一种对ThDP和Mg2 +的亲和力都降低了。据报道，辅因子与脱辅基PDC的结合会淬灭内在的色氨酸荧光[Diefenbach和Duggleby（1991年）《生物化学杂志》276，439 - 445]，并且我们已经确定负责此作用的残基为Trp - 487 [Diefenbach、Candy、Mattick和Duggleby（1992年）《欧洲生物化学学会联合会快报》296，95 - 98]。尽管这个残基距离辅因子结合位点有一段距离，但它位于二聚体界面，并且有人提出[Dyda、Furey、Swaminathan、Sax、Farrenkopf和Jordan（1993年）《生物化学》32，6165 - 6170]，当辅因子结合时，与相邻亚基的Phe - 496的环堆积改变是荧光淬灭的机制。密切相关的酶吲哚丙酮酸脱羧酶（来自阴沟肠杆菌）在对应于Phe - 496的位置有一个亮氨酸残基，但显示出与PDC相似的荧光淬灭特性。这表明荧光淬灭是由于Trp - 487局部环境的某种扰动，而不是与Phe - 496的特异性相互作用。我们的数据支持后一种假设：在PDC中将这个苯丙氨酸突变为亮氨酸、异亮氨酸或组氨酸并不会消除添加辅因子后的荧光淬灭。
