COMPARATIVE ENZYMATIC-PROPERTIES OF GAPB-ENCODED ERYTHROSE-4-PHOSPHATE DEHYDROGENASE OF ESCHERICHIA-COLI AND PHOSPHORYLATING GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE

GapB-encoded protein of Escherichia coli and glyceraldehyde-3-phosphat e dehydrogenase (GAPDH) share more than 40% amino acid identity. Most of the amino acids involved in the binding of cofactor and substrates to GAP;DR are conserved in GapB-encoded protein, This enzyme shows an efficient non-phosphorylating erythrose-4-phosphate dehydrogenase acti vity (Zhao, G., Pease, A, J., Bharani, N., and Winkler, M. E. (1995) J . Bacteriol: 177, 2804-2812) but a low phosphorylating glyceraldehyde- 3-phosphate dehydrogenase activity, whereas GAPDN shows a high efficie nt phosphorylating glyceraldehyde-8-phosphate dehydrogenase activity a nd a low phosphorylating erythrose-4-phosphate dehydrogenase activity, To identify the structural factors responsible for these differences, comparative kinetic and binding studies have been carried out on both GapB-encoded protein of Escherichia coli and GAPDH of Bacillus stearo thermophilus. The K-D constant of GapB-encoded protein for NAD is 800- fold higher than that of GAPDH. The chemical mechanism of erythrose 4- phosphate oxidation by GapB-encoded protein is shown to proceed throug h a two-step mechanism involving covalent intermediates with Cys-149, with rates associated to the acylation and deacylation processes of 28 0 s(-1) and 20 s(-1), respectively. No isotopic solvent effect is obse rved suggesting that the rate-limiting step is not hydrolysis. The rat e of oxidation of glyceraldehyde 3-phosphate is 0.12 s(-1) and is hydr ide transfer limiting, at least 2000-fold less efficient compared with that of erythrose 4-phosphate. Thus, it can be concluded that it is o nly the structure of the substrates that prevails in forming a ternary complex enzyme-NAD-thiohemiacetal productive (or not) for hydride tra nsfer in the acylation step, This conclusion is reinforced by the fact that the rats of oxidation for erythrose 4-phosphate by GAPDH is 0.1 s(-1) and is limited by the acylation. step, whereas glyceraldehyde 3- phosphate acylation is efficient and is not rate-determining (greater than or equal to 800 s(-1)), Substituting Asn for His-176 on GapB-enco ded protein, a residue postulated to facilitate hydride transfer as a base catalyst, decreases 40-fold the k(cat) of glyceraldehyde 8-phosph ate oxidation. This suggests that the non-efficient positioning of the C-1 atom of glyceraldehyde 3-phosphate relative to the pyridinium of the cofactor within the ternary complex is responsible for the low cat alytic efficiency, No phosphorylating activity on erythrose 4-phosphat e with GapB-encoded protein is observed although the Pi site is operat ive as proven by the oxidative phosphorylation of glyceraldehyde 3-pho sphate. Thus the binding of inorganic phosphate to the ki site likely is not productive for attacking efficiently the thioacyl intermediate formed with erythrose 4-phosphate, whereas a water molecule is an effi cient nucleophile for the hydrolysis of the thioacyl intermediate, Com pared with glyceraldehyde-3-phosphate dehydrogenase activity, this cor responds to an activation of the deacylation step by greater than or e qual to 4.5 Kcal.mol(-1). Altogether these results suggest subtle stru ctural differences between the active sites of GAPDH and GapB-encode p rotein that could be revealed and/or modulated by the structure of the substrate bound. This also indicates that a protein engineering appro ach could be used to convert a phosphorylating aldehyde dehydrogenase into an efficient non-phosphorylating one and vice versa.