Lack of asymmetry in the active sites of tetrameric D-glyceraldehyde-3-phosphate dehydrogenase during alkylation in the crystalline state

Abstract

D-Glyceraldehyde-3-phosphate dehydrogenase (GPDH; EC 1.2.1.12) catalyzes the oxidative phosphorylation of D-glyceraldehyde-3-phosphate (GAP) through the formation of an acyl thiol enzyme. It is composed of 4 subunits of identical amino acid sequence (review [I]). The X-ray diffraction studies indicated that the subunit structure of crystalline GPDH from different sources 12-61, except that from Bacillus stearothermophilus [7], displays a pairwise asymmetry which may have functional consequences. In the case of lobster GPDH crystals it was also suggested that Cys-149 bearing the essential thiol group interacted with His-176 only in 2 of the 4 subunits 121. This implies that the mercaptide-imidazolium ion-pair formation between Cys149 and His176, which enhances the nucleophilicity of the thiol group below its pK, 181, is formed in 2 subunits only. Hence, it could be expected that the essential cysteine residues in the crystal may react at different rates in a simple alkylation. It has been shown that all 4 reactive sulphydryl groups of GPDH crystals could be alkylated with the negatively charged iodoacetate 191, but quantitative data on the reaction rates were not presented. mercuribenzoate [ 141 exhibit ‘half-of-the-sites’ reactivity even in solution and thus they are not suitable for studying this problem. Furthermore, with the neutral iodoacetamide, electrostatic effects which may complicate the reaction could also be eliminated. We have found that all 4 subunits of the crystalline enzyme react with iodoacetamide at identical rates and the rates of alkylation are similar in crystal and in solution.