Talfournier F, Colloc'h N, Mornon J P, Branlant G
UMR 7567 CNRS-UHP Maturation des ARN et Enzymologie Moléculaire, Vandoeuvre-les-Nancy, France.
Eur J Biochem. 1998 Mar 15;252(3):447-57. doi: 10.1046/j.1432-1327.1998.2520447.x.
Phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GraP-DH) catalyzes the oxidative phosphorylation of D-glyceraldehyde-3-phosphate to form 1.3-diphosphoglycerate. The currently accepted mechanism involves an oxidoreduction step followed by a phosphorylation. Two essential aminoacids, Cys149 and His176 are involved in the chemical mechanism of bacterial and eukaryotic GraP-DHs. Roles have been assigned to the His176 as (a) a chemical activator for enhancing the reactivity of Cys149, (b) a stabilizator of the tetrahedral transition states, and (c) a base catalyst facilitating hydride transfer towards NAD. In a previous study carried out on Escherichia coli GraP-DH [Soukri, A., Mougin, A., Corbier, C., Wonacott, A. J., Branlant, C. & Branlant, G. (1989) Biochemistry, 28, 2586-2592], the role of His176 as an activator of the reactivity of Cys149 was studied. Here, we further investigated the role of the His residue in the chemical mechanism of phosphorylating GraP-DH from E. coli and Bacillus stearothermophilus. The chemical reactivity of Cys149 in the His176Asn mutant was reinvestigated. At neutral pH, its reactivity was shown to be at least as high as that observed in the Cys-/His+ ion pair present in the wild type. No pre-steady state burst of NADH was found with the His176Asn mutant in contrast to what is observed for the wild type, and a primary isotope effect was observed when D-[1-2H]glyceraldehyde-3-phosphate was used as the substrate. Therefore, the major role of the His176 in the catalytic mechanism under physiological conditions is not to activate the nucleophilicity of Cys149 but first to facilitate the hydride transfer. These results hypothesized that a phosphorylating GraP-DH possessing a different protein environment competent to increase the nucleophilic character of the essential Cys residue and to favor the hydride transfer in place of His, could be enzymically efficient. This is most likely the case for archaeal Methanothermus fervidus GraP-DH which shares less than 15% amino-acid identity with the bacterial or eukaryotic counterparts. No Cys-/His+ ion pair was detectable. Only one thiolate entity was observed with an apparent pKa of 6.2. This result was confirmed by the fact that none of the mutations of the five invariant His changed the catalytic efficiency.
磷酸化甘油醛 - 3 - 磷酸脱氢酶（GAP - DH）催化D - 甘油醛 - 3 - 磷酸的氧化磷酸化反应，生成1,3 - 二磷酸甘油酸。目前被广泛接受的机制是先进行氧化还原步骤，然后是磷酸化反应。两个必需氨基酸，半胱氨酸149（Cys149）和组氨酸176（His176）参与了细菌和真核生物GAP - DH的化学机制。His176的作用被认为有：（a）作为化学活化剂，增强Cys149的反应活性；（b）作为四面体过渡态的稳定剂；（c）作为促进氢化物向NAD转移的碱催化剂。在之前对大肠杆菌GAP - DH进行的一项研究中[Soukri, A., Mougin, A., Corbier, C., Wonacott, A. J., Branlant, C. & Branlant, G. (1989) Biochemistry, 28, 2586 - 2592]，研究了His176作为Cys149反应活性活化剂的作用。在此，我们进一步研究了His残基在大肠杆菌和嗜热栖热芽孢杆菌磷酸化GAP - DH化学机制中的作用。重新研究了His176Asn突变体中Cys149的化学反应活性。在中性pH条件下，其反应活性至少与野生型中存在的Cys⁻/His⁺离子对所观察到的反应活性一样高。与野生型不同，His176Asn突变体未发现NADH的预稳态爆发，并且当使用D - [1 - ²H]甘油醛 - 3 - 磷酸作为底物时观察到了初级同位素效应。因此，在生理条件下His176在催化机制中的主要作用不是激活Cys149的亲核性，而是首先促进氢化物转移。这些结果推测，具有不同蛋白质环境、能够增强必需半胱氨酸残基的亲核特性并有利于替代His进行氢化物转移的磷酸化GAP - DH，可能具有酶促效率。嗜热栖热芽孢杆菌的古菌GAP - DH很可能就是这种情况，它与细菌或真核生物的对应物氨基酸序列同一性不到15%。未检测到Cys⁻/His⁺离子对。仅观察到一个硫醇盐实体，其表观pKa为6.2。五个不变His的突变均未改变催化效率这一事实证实了该结果。
