Comparative Enzymatic Properties of GapB-encoded Erythrose-4-Phosphate Dehydrogenase of Escherichia coliand Phosphorylating Glyceraldehyde-3-phosphate Dehydrogenase
1997; Elsevier BV; Volume: 272; Issue: 24 Linguagem: Inglês
10.1074/jbc.272.24.15106
ISSN1083-351X
AutoresSandrine Boschi‐Müller, Saı̈d Azza, David Pollastro, Catherine Corbier, Guy Branlant,
Tópico(s)Mass Spectrometry Techniques and Applications
ResumoGapB-encoded protein of Escherichia coli and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) share more than 40% amino acid identity. Most of the amino acids involved in the binding of cofactor and substrates to GAPDH are conserved in GapB-encoded protein. This enzyme shows an efficient non-phosphorylating erythrose-4-phosphate dehydrogenase activity (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 GAPDH shows a high efficient phosphorylating glyceraldehyde-3-phosphate dehydrogenase activity and 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 stearothermophilus. TheK 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 through a two-step mechanism involving covalent intermediates with Cys-149, with rates associated to the acylation and deacylation processes of 280 s−1 and 20 s−1, respectively. No isotopic solvent effect is observed suggesting that the rate-limiting step is not hydrolysis. The rate of oxidation of glyceraldehyde 3-phosphate is 0.12 s−1 and is hydride transfer limiting, at least 2000-fold less efficient compared with that of erythrose 4-phosphate. Thus, it can be concluded that it is only the structure of the substrates that prevails in forming a ternary complex enzyme-NAD-thiohemiacetal productive (or not) for hydride transfer in the acylation step. This conclusion is reinforced by the fact that the rate 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 (≥800 s−1). Substituting Asn for His-176 on GapB-encoded protein, a residue postulated to facilitate hydride transfer as a base catalyst, decreases 40-fold thek cat of glyceraldehyde 3-phosphate 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 catalytic efficiency. No phosphorylating activity on erythrose 4-phosphate with GapB-encoded protein is observed although the Pi site is operative as proven by the oxidative phosphorylation of glyceraldehyde 3-phosphate. Thus the binding of inorganic phosphate to the Pi site likely is not productive for attacking efficiently the thioacyl intermediate formed with erythrose 4-phosphate, whereas a water molecule is an efficient nucleophile for the hydrolysis of the thioacyl intermediate. Compared with glyceraldehyde-3-phosphate dehydrogenase activity, this corresponds to an activation of the deacylation step by ≥4.5 kcal·mol−1. Altogether these results suggest subtle structural differences between the active sites of GAPDH and GapB-encoded protein that could be revealed and/or modulated by the structure of the substrate bound. This also indicates that a protein engineering approach could be used to convert a phosphorylating aldehyde dehydrogenase into an efficient non-phosphorylating one andvice versa. GapB-encoded protein of Escherichia coli and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) share more than 40% amino acid identity. Most of the amino acids involved in the binding of cofactor and substrates to GAPDH are conserved in GapB-encoded protein. This enzyme shows an efficient non-phosphorylating erythrose-4-phosphate dehydrogenase activity (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 GAPDH shows a high efficient phosphorylating glyceraldehyde-3-phosphate dehydrogenase activity and 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 stearothermophilus. TheK 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 through a two-step mechanism involving covalent intermediates with Cys-149, with rates associated to the acylation and deacylation processes of 280 s−1 and 20 s−1, respectively. No isotopic solvent effect is observed suggesting that the rate-limiting step is not hydrolysis. The rate of oxidation of glyceraldehyde 3-phosphate is 0.12 s−1 and is hydride transfer limiting, at least 2000-fold less efficient compared with that of erythrose 4-phosphate. Thus, it can be concluded that it is only the structure of the substrates that prevails in forming a ternary complex enzyme-NAD-thiohemiacetal productive (or not) for hydride transfer in the acylation step. This conclusion is reinforced by the fact that the rate 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 (≥800 s−1). Substituting Asn for His-176 on GapB-encoded protein, a residue postulated to facilitate hydride transfer as a base catalyst, decreases 40-fold thek cat of glyceraldehyde 3-phosphate 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 catalytic efficiency. No phosphorylating activity on erythrose 4-phosphate with GapB-encoded protein is observed although the Pi site is operative as proven by the oxidative phosphorylation of glyceraldehyde 3-phosphate. Thus the binding of inorganic phosphate to the Pi site likely is not productive for attacking efficiently the thioacyl intermediate formed with erythrose 4-phosphate, whereas a water molecule is an efficient nucleophile for the hydrolysis of the thioacyl intermediate. Compared with glyceraldehyde-3-phosphate dehydrogenase activity, this corresponds to an activation of the deacylation step by ≥4.5 kcal·mol−1. Altogether these results suggest subtle structural differences between the active sites of GAPDH and GapB-encoded protein that could be revealed and/or modulated by the structure of the substrate bound. This also indicates that a protein engineering approach could be used to convert a phosphorylating aldehyde dehydrogenase into an efficient non-phosphorylating one andvice versa. The glycolytic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) 1The abbreviations used are: GAPDH,d-glyceraldehyde-3-phosphate dehydrogenase; G3P, glyceraldehyde 3-phosphate; 1,3-dPG, 1,3-diphosphoglycerate; E4P, erythrose 4-phosphate; DTNB, 5,5′-dithiobis-(2-nitrobenzoic acid); TES,N-tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid.1The abbreviations used are: GAPDH,d-glyceraldehyde-3-phosphate dehydrogenase; G3P, glyceraldehyde 3-phosphate; 1,3-dPG, 1,3-diphosphoglycerate; E4P, erythrose 4-phosphate; DTNB, 5,5′-dithiobis-(2-nitrobenzoic acid); TES,N-tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid. is a tetrameric enzyme that catalyzes reversibly the oxidative phosphorylation ofd-glyceraldehyde 3-phosphate (G3P) to form 1,3-diphosphoglycerate (1,3-dPG) in the presence of NAD and inorganic phosphate (1Harris J.I. Waters M. Boyer P.D. The Enzymes. 13. Academic Press, New York1976: 1-49Google Scholar). The refined structures of several phosphorylating GAPDHs have already been reported (2Moras D. Olsen K.W. Sabesan M.N. Buehner M. Ford G.C. Rossmann M.G. J. Biol. Chem. 1975; 250: 9137-9162Abstract Full Text PDF PubMed Google Scholar, 3Skarzynski T. Moody P.C.E. Wonacott A.J. J. Mol. Biol. 1987; 193: 171-187Crossref PubMed Scopus (286) Google Scholar, 4Korndörfer I. Steipe B. Huber R. Tomschy A. Jaenicke R. J. Mol. Biol. 1995; 246: 511-521Crossref PubMed Scopus (225) Google Scholar, 5Kim H. Feil I.K. Verlinde C.L.M.J. Petra P.H. Hol W.G.J. Biochemistry. 1995; 34: 14975-14986Crossref PubMed Scopus (109) Google Scholar, 6Tanner J.J. Hecht R.M. Krause K.L. Biochemistry. 1996; 35: 2597-2609Crossref PubMed Scopus (208) Google Scholar, 7Duée E. Olivier-Deyris L. Fanchon E. Corbier C. Branlant G. Dideberg O. J. Mol. Biol. 1996; 257: 814-838Crossref PubMed Scopus (89) Google Scholar). The currently accepted forward reaction pathway involves two steps, first the formation of a covalent ternary complex GAPDH-NAD-G3P preceding an oxidoreduction step that leads to a thioacyl enzyme intermediate and NADH, and second the phosphorylation that produces 1,3-dPG. The chemical mechanism of catalysis was extensively studied and is now well understood (8Soukri A. Mougin A. Corbier C. Wonacott A.J. Branlant C. Branlant G. Biochemistry. 1989; 28: 2586-2592Crossref PubMed Scopus (69) Google Scholar, 9Corbier C. Della Seta F. Branlant G. Biochemistry. 1992; 31: 12532-12535Crossref PubMed Scopus (24) Google Scholar). The structural determinants of the nicotinamide and adenosine subsites involved in coenzyme specificity that control the catalytic efficiency have been recently analyzed (10Corbier C. Clermont S. Billard P. Skarzynski T. Branlant C. Wonacott A. Branlant G. Biochemistry. 1990; 29: 7016-7101Crossref Scopus (50) Google Scholar, 11Corbier C. Mougin A. Mely Y. Adolph H.W. Zeppezauer M. Gerard D. Wonacott A. Branlant G. Biochimie ( Paris ). 1990; 72: 545-554Crossref PubMed Scopus (29) Google Scholar, 12Clermont S. Corbier C. Mely Y. Gerard D. Wonacott A. Branlant G. Biochemistry. 1993; 32: 10178-10184Crossref PubMed Scopus (74) Google Scholar, 13Eyschen J. Vitoux B. Rahuel-Clermont S. Marraud M. Branlant G. Cung M.T. Biochemistry. 1996; 35: 6064-6072Crossref PubMed Scopus (12) Google Scholar). The individual contribution of the amino acids implicated in the two phosphate binding sites named Ps and Pi sites has also been studied at the kinetic, structural, and energetic levels (14Corbier C. Michels S. Wonacott A. Branlant G. Biochemistry. 1994; 33: 3260-3265Crossref PubMed Scopus (32) Google Scholar, 15Michels S. Rogalska E. Branlant G. Eur. J. Biochem. 1996; 235: 641-647Crossref PubMed Scopus (22) Google Scholar).GAPDH is a key enzyme of the glycolysis and gluconeogenesis pathways. In the gram− Escherichia coli, three distinct gap genes have been isolated so far. The gapA gene was shown to encode an efficient active GAPDH (16Branlant G. Branlant C. Eur. J. Biochem. 1985; 150: 61-66Crossref PubMed Scopus (121) Google Scholar, 17Charpentier B. Branlant C. J. Bacteriol. 1994; 176: 830-839Crossref PubMed Google Scholar). A second gapgene, called gapB, was initially characterized by Alefounder and Perham (18Alefounder P.R. Perham R.N. Mol. Microbiol. 1989; 3: 723-732Crossref PubMed Scopus (86) Google Scholar). This gene belongs to a cluster of genes coding for several glycolytic enzymes. This gene organization is often found in eubacteria and archaebacteria studied so far (Ref. 17Charpentier B. Branlant C. J. Bacteriol. 1994; 176: 830-839Crossref PubMed Google Scholar and references cited therein) and is usually responsible for the GAPDH activity. However, a peculiar situation is observed in E. coli. ThegapB gene, which is located within the glycolytic gene cluster, does not seem to encode GAPDH activity (18Alefounder P.R. Perham R.N. Mol. Microbiol. 1989; 3: 723-732Crossref PubMed Scopus (86) Google Scholar). Indeed, Hillman and Fraenkel (19Hillman J.D. Fraenkel D.G. J. Bacteriol. 1975; 122: 1175-1179Crossref PubMed Google Scholar) showed that a nonsense mutation in thegapA gene abolishes GAPDH activity. Thus, the GAPDH activity is only encoded by the gapA gene that is 22.6 min far from the phosphoglycerate kinase gene on the E. coli chromosome. This has led to the proposal of either an eukaryotic origin for thegapA gene (18Alefounder P.R. Perham R.N. Mol. Microbiol. 1989; 3: 723-732Crossref PubMed Scopus (86) Google Scholar, 20Martin W. Cerff R. Eur. J. Biochem. 1986; 159: 323-331Crossref PubMed Scopus (99) Google Scholar, 21Doolittle R.F. Feng D.F. Anderson K.L. Alberro M.R. J. Mol. Evol. 1990; 31: 383-388Crossref PubMed Scopus (117) Google Scholar) or the presence of bothgapA and gapB genes in ancestor bacteria (22Martin W. Brinkmann H. Savona C. Cerff R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8692-8696Crossref PubMed Scopus (159) Google Scholar). A third gene named gapC was recently characterized. 2E. Hidalgo, A. Limon, and J. Aguilar, unpublished results.2E. Hidalgo, A. Limon, and J. Aguilar, unpublished results. The presence of several stop codons in the putative coding regions, clearly indicates thatgapC gene can not encode a functional GAPDH.Amino acid sequence comparison of GAPDHs from E. coli andBacillus stearothermophilus and GapB 3To avoid misunderstanding, GAPDH refers to protein having high activity with G3P. The protein encoded by thegapB gene, which showed low GAPDH activity, is named GapB-encoded protein.3To avoid misunderstanding, GAPDH refers to protein having high activity with G3P. The protein encoded by thegapB gene, which showed low GAPDH activity, is named GapB-encoded protein.-encoded protein shows 41.6 and 43.6% identity, respectively. Nearly all the amino acids essential for the chemical mechanism and for the binding of cofactor and substrates are conserved. In the present paper, the enzymatic properties of the protein encoded by the gapB gene are described and compared with those of the GAPDH from B. stearothermophilus. The results are discussed in relation to the three-dimensional structures of the E. coli (7Duée E. Olivier-Deyris L. Fanchon E. Corbier C. Branlant G. Dideberg O. J. Mol. Biol. 1996; 257: 814-838Crossref PubMed Scopus (89) Google Scholar) and B. stearothermophilus (3Skarzynski T. Moody P.C.E. Wonacott A.J. J. Mol. Biol. 1987; 193: 171-187Crossref PubMed Scopus (286) Google Scholar) GAPDHs and to the recent data of Zhao et al. (23Zhao G. Pease A.J. Bharani N. Winkler M.E. J. Bacteriol. 1995; 177: 2804-2812Crossref PubMed Scopus (74) Google Scholar), who showed that GapB-encoded protein displays an efficient non-phosphorylating erythrose-4-phosphate dehydrogenase activity.RESULTSJustification of the MutationsSite-directed mutagenesis at positions 32, 149, 176, 179, 206–209, and 311 were done for the following reasons (see also Fig.1).Positions 149 and 176These positions are always occupied by a Cys and a His residue, respectively, in all GAPDHs described so far (see Fig. 1). Cys-149 forms a thioacyl intermediate during the oxidoreduction process, and His-176 is postulated to decrease the pK a of Cys-149 to favor the hydride transfer and to stabilize the different intermediates formed along the catalytic process (Ref. 8Soukri A. Mougin A. Corbier C. Wonacott A.J. Branlant C. Branlant G. Biochemistry. 1989; 28: 2586-2592Crossref PubMed Scopus (69) Google Scholar and references cited therein). These positions were mutated to check if they could play a similar role during the E4P oxidation process.Position 311This position is always occupied by a Tyr residue in all GAPDHs described so far except for the GapB-encoded protein where a Cys residue is present. Tyr-311 is at the hinge between catalytic and cofactor domains and is located near the catalytic amino acids Cys-149 and His-176 and also near Cys-153 (see Fig. 7 in Ref. 7). Modeling cannot exclude the possibility for Cys-311 to form an alternative thioacyl enzyme intermediate with E4P that has an additional CHOH group compared with G3P.Position 179This position belongs to the Ps site and is always occupied by a Thr in all active GAPDHs described so far (15Michels S. Rogalska E. Branlant G. Eur. J. Biochem. 1996; 235: 641-647Crossref PubMed Scopus (22) Google Scholar) except for the GapB-encoded protein where a Met residue is present (18Alefounder P.R. Perham R.N. Mol. Microbiol. 1989; 3: 723-732Crossref PubMed Scopus (86) Google Scholar).Position 32This position is always occupied by an Asp residue, which forms a hydrogen bond with both 2′- and 3′- hydroxyl groups of the ribose of the adenosine moiety, except for the GapB-encoded protein where a Glu residue is present. Since the substitution Asp-32 → Glu in B. stearothermophilus GAPDH increased 9-fold the K M of NAD (12Clermont S. Corbier C. Mely Y. Gerard D. Wonacott A. Branlant G. Biochemistry. 1993; 32: 10178-10184Crossref PubMed Scopus (74) Google Scholar), the presence of a Glu-32 could be one of the factors responsible for the low affinity of NAD(H) of the GapB-encoded protein (see “Results”).Position 206–209This sequence contained positions always invariant in GAPDH that are involved in the Pi site, i.e.Thr-208 and Gly-209. This sequence is largely changed in the GapB-encoded protein, where in particular Gly-209 is substituted by a Lys residue (see Fig. 1).Biochemical Properties of Wild-type and Mutated GapB-encoded ProteinGapB-encoded protein was overexpressed in E. colistrain using a plasmidic construction containing the gapBgene under either its own promoter or the gapApromoter.4 Over 15% of the soluble proteins in the supernatant were GapB-encoded proteins. The protocol used to purify GapB-encoded protein to homogeneity took advantage of the higher hydrophobic character of GapB-encoded protein compared with the GAPDH from E. coli. This allowed easy separation of GapB-encoded protein from GAPDH on a hydrophobic column. This protocol is different from that used by Zhao et al. (23Zhao G. Pease A.J. Bharani N. Winkler M.E. J. Bacteriol. 1995; 177: 2804-2812Crossref PubMed Scopus (74) Google Scholar) (see TableI). The molecular weight of 37,170 ± 2 determined by mass spectrometry was in agreement with the 37,169 mass predicted from the gapB DNA sequence but is different to that calculated by Zhao et al. (23Zhao G. Pease A.J. Bharani N. Winkler M.E. J. Bacteriol. 1995; 177: 2804-2812Crossref PubMed Scopus (74) Google Scholar). The fact that GapB-encoded protein precipitated at low ammonium sulfate concentration (45% instead of 66% for all GAPDHs) and migrated faster in SDS-polyacrylamide gel than expected from its molecular weight compared with the GAPDH from E. coli and B. stearothermophilus (molecular weight of 35,401 and 35,944, respectively, gel not shown) is certainly related to its hydrophobic character. The protein was isolated as apo form, as judged by the ratioA 280/A 260 of 2. The presence of a reducing agent along the protocol of purification and during the storage of the purified enzyme had no effect on the activity in contrast to what Zhao et al. (23Zhao G. Pease A.J. Bharani N. Winkler M.E. J. Bacteriol. 1995; 177: 2804-2812Crossref PubMed Scopus (74) Google Scholar) have observed.Table IPurification of GapB-encoded proteinFractionTotal proteinSpecific activityTotal activityPurification factorYieldmgμmol · min−1 · mg−1μmol · min−1(-fold)%Homogenate89570.1412791100(NH4)2SO439470.271066283ACA 343632.348491766Q-Sepharose5214.3074210258Phenyl-Sepharose5014.7073510557E4PDH activity was determined at 25 °C in 40 mmtriethanolamine buffer. 0.2 mm EDTA, pH 8.9, using nonpurified E4P as a substrate. Concentrations of NAD and E4P were 2 mm. Open table in a new tab Thiol titrations by DTNB showed four titratable cysteines per monomer under denaturing conditions. This result is expected from thegapB DNA sequence that indicates cysteine residues at positions 95, 149, 153, and 311. Two cysteine residues showing different reactivity were titratable per monomer under native conditions, with one having a high reactivity compared with that of the second one. This suggested that, in addition to the Cys-149 that was shown to be accessible and highly reactive in GAPDH (8Soukri A. Mougin A. Corbier C. Wonacott A.J. Branlant C. Branlant G. Biochemistry. 1989; 28: 2586-2592Crossref PubMed Scopus (69) Google Scholar), a second cysteine residue was also titratable. The fact that two cysteine residues were titratable for C95S and C153S mutants and that only one cysteine was titratable for C311A, S and Y mutants indicates that Cys-311 is the second titratable cysteine.By combining stopped-flow and classical spectrophotometry experiments,k obs values of 136 s−1 and 8.10−3 s−1 for wild type and of 116 s−1 for C311A mutant were determined in the absence of NAD. This proved that Cys-149 is the most reactive titratable cysteine within the active site. A k obs value of 242 s−1 was found for the titratable Cys-149 of the GAPDH ofB. stearothermophilus. Thus, the Cys-149 of GapB-encoded protein has a chemical reactivity in the range of that observed for GAPDH. It is noteworthy that in C149A, G, V mutants, the Cys-311 was not titratable.KineticsWith G3P as a SubstrateThe kinetic parameters (k cat and K M) of GapB-encoded protein and its various mutants are summarized in TableII. A 600-fold decrease in thek cat value in the forward reaction was observed for the GapB-encoded protein compared with the GAPDH from B. stearothermophilus. K M value for NAD increased 10-fold. To determine the nature of the limiting step and to define whether the catalytic mechanism proceeded via two distinct chemical steps as shown for GAPDH, transient kinetics in the absence or presence of inorganic phosphate (50 mm) were carried out at saturating NAD concentrations. Under these experimental conditions no burst of NADH production was observed. The rate constant of 0.12 s−1 is similar to the k cat value obtained under steady state conditions regardless of the presence or absence of inorganic phosphate (curves not shown). This demonstrated that the limiting step is associated with the formation of the acyl enzyme intermediate and not with steps occurring after the acylation, as shown for the GAPDHs from E. coli and B. stearothermophilus (11Corbier C. Mougin A. Mely Y. Adolph H.W. Zeppezauer M. Gerard D. Wonacott A. Branlant G. Biochimie ( Paris ). 1990; 72: 545-554Crossref PubMed Scopus (29) Google Scholar, 14Corbier C. Michels S. Wonacott A. Branlant G. Biochemistry. 1994; 33: 3260-3265Crossref PubMed Scopus (32) Google Scholar). The foregoing results indicated an acylation step at least 7000-fold less efficient compared with the GAPDH from B. stearothermophilus (15Michels S. Rogalska E. Branlant G. Eur. J. Biochem. 1996; 235: 641-647Crossref PubMed Scopus (22) Google Scholar). The presence of an isotopic effect of 5 with d-[1-2H]G3P as a substrate (data not shown) demonstrated that the rate-limiting step is associated with the hydride transfer.Table IIKinetic parameters of wild-type and mutants of GapB-encoded protein, and wild-type and mutant of B. stearothermophilus GAPDHSubstrateG3PE4PConstantKM G3PKM NADk catKM E4PKM NADk catmmmms−1mmmms−1Wild-type GapB-encoded protein1.10 ± 0.500.90 ± 0.200.12 ± 0.040.51 ± 0.090.8 ± 0.220 ± 1E32D mutant1.80 ± 0.201.10 ± 0.200.10 ± 0.051.90 ± 0.202.3 ± 0.47.4 ± 0.4H176N mutant3.00 ± 1.000.73 ± 0.040.0030 ± 0.00060.24 ± 0.040.5 ± 0.10.21 ± 0.01M179T mutant1.00 ± 0.401.00 ± 0.200.11 ± 0.030.70 ⊥ 0.103.5 ± 0.64.0 ± 0.4C311A mutant0.41 ± 0.081.00 + 0.100.035 ± 0.0030.80 ± 0.104 ± 13.5 ± 0.5C311Y mutant0.30 ± 0.102.20 ± 0.700.020 ± 0.0030.70 ± 0.305 ± 20.6 ± 0.1Wild-type B. st.0.90 ± 0.200.09 ± 0.0176 ± 43.30 ± 1.100.08 ± 0.020.10 ± 0.02GAPDHT179M mutant2-aFrom Michels et al. (15).0.16 ± 0.030.31 ± 0.050.015 ± 0.005NDNDNDGAPDH activity was determined at 25 °C in 40 mmtriethanolamine buffer, 0.2 mm EDTA, and 50 mmK2HPO4, pH 8.9, for the oxidative phosphorylation and in 25 mm imidazole buffer pH 7, for the reverse reaction.KM values are the average of two independent determinations. E4PDH activity was determined at 25 °C in 40 mm triethanolamine buffer, 0.2 mm EDTA, pH 8.9, using purified E4P as described under “Materials and Methods.” ND, not determined. B. st., B. stearothermophilus.2-a From Michels et al. (15Michels S. Rogalska E. Branlant G. Eur. J. Biochem. 1996; 235: 641-647Crossref PubMed Scopus (22) Google Scholar). Open table in a new tab The fact that acylation was limiting did not exclude an efficient phosphorylating step. To investigate this possibility, the chemical nature of the product formed in the forward direction in the absence and presence of 0.1 m phosphate was determined enzymatically and characterized chemically by 31P NMR. It was shown to be 3-phosphoglycerate and 1,3-dPG, respectively (see “Materials and Methods”). Thus, the GapB-encoded protein exhibits a phosphorylating activity more efficient than the non-phosphorylating one. Clearly, the Pi site is operational. In the reverse direction, 1,3-dPG was shown to be a substrate with a K M value increased 16-fold, a K M value of NADH increased 5-fold, and a k cat decreased 880-fold compared with GAPDH (K M (1,3-dPG) 0.08 and 0.005 mm, K M (NADH) 0.05 and 0.011 mm and k cat 0.074 s−1and 65 s−1 for GapB-encoded protein and GAPDH of B. stearothermophilus, respectively).As shown in Table II, replacing Met-179 by Thr and Cys-311 by Tyr or Ala did not drastically change the GAPDH activity of GapB-encoded protein, whereas changing His-176 into Asn decreasedk cat by a factor of 40. No significant activity was observed for C149G, A or V mutants under the experimental conditions used (data not shown, see “Discussion”).With E4P as a SubstrateThe data confirmed those already published by Zhao et al. (23Zhao G. Pease A.J. Bharani N. Winkler M.E. J. Bacteriol. 1995; 177: 2804-2812Crossref PubMed Scopus (74) Google Scholar) showing an E4P dehydrogenase activity. A k cat of 20 s−1 and aK M of 800 μm for NAD were found under optimal conditions. The k cat value is 2.5-fold smaller than that described previously (23Zhao G. Pease A.J. Bharani N. Winkler M.E. J. Bacteriol. 1995; 177: 2804-2812Crossref PubMed Scopus (74) Google Scholar). This discrepancy remains to be explained. An E4P dehydrogenase activity for C149G, A and V mutants, purified from a E. coli strain, was observed at least 2000-fold less compared with wild type. This low activity is in fact due to the GapB-encoded protein expressed from the chromosomicgapB gene of E. coli strain used to overexpress the mutants. Indeed the mutants purified from a ΔgapBdeleted strain showed no significant activity, at least 106-fold less compared with wild type (data not shown). The fact that CD spectra of the mutants and wild type perfectly superposed (curves not shown) suggests that the absence of activity of the mutants is not a consequence of change in their structure. Replacing amino acids located in the catalytic subsite, i.e. Met-179 by Thr, Cys-311 by Ala or Tyr, His-176 by Asn reducedk cat by a factor of 5, 6, 33, and 95, respectively.Pre-steady state stopped-flow experiments showed a burst of 1 mol of NADH production per monomer with a k obs of 230 and 280 s−1 at pH 8.2 and 8.9, respectively (curves not shown). This supported a two-step mechanism with formation of a thioacyl intermediate involving Cys-149 and a rate-limiting step occurring after NADH formation. This limiting step could either be the deacylation step or the release of 4-phosphoerythronate or NADH that could include an isomerization step. The fact that no D2O isotopic effect (data not shown) was observed rather excluded the first hypothesis. The kinetics are similar in the presence of up to 100 mm phosphate. The fact that the deacylation step is not rate-limiting does not exclude that phosphorylation can occur with an efficiency similar or even higher than deacylation. Two kinds of results argue, however, against this possibility. First, erythrose 4-phosphate oxidation proceeded to completion in the presence or absence of 50 mm phosphate, as measured from NADH production (data not shown), whereas, based on the equilibrium constant of the oxidative phosphorylation of G3P (K eq = 2.10−8 (29Canellas P.F. Cleland W.W. Biochemistry. 1991; 30: 8871-8876Crossref PubMed Scopus (16) Google Scholar)), the oxidative phosphorylation of E4P was expected to yield only 56% 1,4-diphosphoerythronate under the experimental conditions used (0.1 mm E4P, 0.1 mm NAD, 40 mm triethanolamine buffer, 2 mm EDTA, 50 mm phosphate, pH 8.9). Second, only 4-phosphoerythronate is formed as characterized by 31P NMR (see “Materials and Methods”).The catalytic efficiency of the GapB-encoded protein tested with erythrose is highly decreased (k cat 0.36 s−1, K M (erythrose) 90 mm). This points out the role of the phosphate at the C-4 position for revealing the erythrose dehydrogenase activity.E4P is also a substrate for GAPDH from B. stearothermophilus. k cat is reduced 760- and 200-fold when compared with that of G3P activity of GAPDH and E4P activity of GapB-encoded protein, respectively (see Table II). This activity is of phosphorylating type as judged by the yield of NADH production. 1,4-Diphosphoerythronate was isolated using a method similar to that used for 1,3-dPG (9Corbier C. Della Seta F. Branlant G. Biochemistry. 1992; 31: 12532-12535Crossref PubMed Scopus (24) Google Scholar) and characterized enzymatically using the reverse reaction of GAPDH. Transient kinetics in the absence or presence of inorganic phosphate (50 mm) showed no burst of NADH production, with ak obs similar to the k catvalue (curves not shown). This showed that the limiting step is associated to the acyl enzyme formation.NAD Binding to GapB-encoded ProteinTo determine whether the higher K M value for NAD is indicative of a decrease of its affinity, K D value was determined using iodoacetamide as a second order labeling probe of cysteine by measuring the protection against inactivation afforded by the coenzyme binding. The deduced K D value provides a measure of mac
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