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W2084566211 abstract "In order to address the molecular basis of the specificity of aldehyde dehydrogenase for aldehyde substrates, enzymatic characterization of the glyceraldehyde 3-phosphate (G3P) binding site of non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN) fromStreptococcus mutans has been undertaken. In this work, residues Arg-124, Tyr-170, Arg-301, and Arg-459 were changed by site-directed mutagenesis and the catalytic properties of GAPN mutants investigated. Changing Tyr-170 into phenylalanine induces no major effect on k cat and K m ford-G3P in both acylation and deacylation steps. Substitutions of Arg-124 and Arg-301 by leucine and Arg-459 by isoleucine led to distinct effects on K m, onk cat, or on both. The rate-limiting step of the R124L GAPN remains deacylation. Pre-steady-state analysis and substrate isotope measurements show that hydride transfer remains rate-determining in acylation. Only the apparent affinity ford-G3P is decreased in both acylation and deacylation steps. Substitution of Arg-459 by isoleucine leads to a drastic effect on the catalytic efficiency by a factor of 105. With this R459L GAPN, the rate-limiting step is prior to hydride transfer, and theK m of d-G3P is increased by at least 2 orders of magnitude. Binding of NADP leads to a time-dependent formation of a charge transfer transition at 333 nm between the pyridinium ring of NADP and the thiolate of Cys-302, which is not observed with the holo-wild type. Accessibility of Cys-302 is shown to be strongly decreased within the holostructure. The substitution of Arg-301 by leucine leads to an even more drastic effect with a change of the rate-limiting step similar to that observed for R459I GAPN. Taking into account the three-dimensional structure of GAPN from S. mutans and the data of the present study, it is proposed that 1) Tyr-170 is not essential for the catalytic event, 2) Arg-124 is only involved in stabilizing d-G3P binding via an interaction with the C-3 phosphate, and 3) Arg-301 and Arg-459 participate not only in d-G3P binding via interaction with C-3 phosphate but also in positioning efficientlyd-G3P relative to Cys-302 within the ternary complex GAPN·NADP·d-G3P. In order to address the molecular basis of the specificity of aldehyde dehydrogenase for aldehyde substrates, enzymatic characterization of the glyceraldehyde 3-phosphate (G3P) binding site of non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN) fromStreptococcus mutans has been undertaken. In this work, residues Arg-124, Tyr-170, Arg-301, and Arg-459 were changed by site-directed mutagenesis and the catalytic properties of GAPN mutants investigated. Changing Tyr-170 into phenylalanine induces no major effect on k cat and K m ford-G3P in both acylation and deacylation steps. Substitutions of Arg-124 and Arg-301 by leucine and Arg-459 by isoleucine led to distinct effects on K m, onk cat, or on both. The rate-limiting step of the R124L GAPN remains deacylation. Pre-steady-state analysis and substrate isotope measurements show that hydride transfer remains rate-determining in acylation. Only the apparent affinity ford-G3P is decreased in both acylation and deacylation steps. Substitution of Arg-459 by isoleucine leads to a drastic effect on the catalytic efficiency by a factor of 105. With this R459L GAPN, the rate-limiting step is prior to hydride transfer, and theK m of d-G3P is increased by at least 2 orders of magnitude. Binding of NADP leads to a time-dependent formation of a charge transfer transition at 333 nm between the pyridinium ring of NADP and the thiolate of Cys-302, which is not observed with the holo-wild type. Accessibility of Cys-302 is shown to be strongly decreased within the holostructure. The substitution of Arg-301 by leucine leads to an even more drastic effect with a change of the rate-limiting step similar to that observed for R459I GAPN. Taking into account the three-dimensional structure of GAPN from S. mutans and the data of the present study, it is proposed that 1) Tyr-170 is not essential for the catalytic event, 2) Arg-124 is only involved in stabilizing d-G3P binding via an interaction with the C-3 phosphate, and 3) Arg-301 and Arg-459 participate not only in d-G3P binding via interaction with C-3 phosphate but also in positioning efficientlyd-G3P relative to Cys-302 within the ternary complex GAPN·NADP·d-G3P. nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase aldehyde dehydrogenase glyceraldehyde 3-phosphate 2-(N-morpholino)aminoethane sulfonic acid N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid 2,2′-dipyridyl disulfide ammonium sulfate Nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN)1 catalyzes the irreversible oxidation of d-glyceraldehyde 3-phosphate (d-G3P) into 3-phosphoglycerate in the presence of NADP via a two-step chemical mechanism. It belongs to the aldehyde dehydrogenase (ALDH) superfamily, which oxidizes a wide variety of aldehydes into nonactivated or SCoA-activated acidic compounds (1Lindahl R. Crit. Rev. Biochem. Mol. Biol. 1992; 27: 283-335Crossref PubMed Scopus (360) Google Scholar, 2Vasiliou V. Pappa A. Petersen D.R. Chem. Biol. Interact. 2000; 129: 1-19Crossref PubMed Scopus (298) Google Scholar, 3Vasiliou V. Pappa A. Pharmacology. 2000; 61: 192-198Crossref PubMed Scopus (134) Google Scholar). These enzymes play parts at several levels of cellular metabolism including anabolic and catabolic pathways, detoxification processes, and embryogenesis development. Their numerous functions probably explain why most ALDHs show a wide substrate specificity, except for the CoA-dependent ALDH. In mammals, ALDH class 1 shows preference for retinal but it can also oxidize aromatic and “long-chain” aldehydes with dissimilar catalytic efficiencies, whereas ALDHs that belong to class 2 are more specific for acetaldehyde and short-chain aldehydes (4Klyosov A.A. Rashkovetsky L.G. Tahir M.K. Keung W.M. Biochemistry. 1996; 35: 4445-4456Crossref PubMed Scopus (155) Google Scholar, 5Klyosov A.A. Biochemistry. 1996; 35: 4457-4467Crossref PubMed Scopus (126) Google Scholar). Inspection of the active site of the ALDH structures (i.e. rat dimeric class 3 ALDH, bovine mitochondrial ALDH, sheep liver cytosolic ALDH, rat retinal dehydrogenase type II, cod liver betaine ALDH, human liver mitochondrial ALDH, ALDH from Vibrio harveyi, and GAPN fromThermoproteus tenax (6Liu Z.J. Sun Y.J. Rose J. Chung Y.J. Hsiao C.D. Chang W.R. Kuo I. Perozich J. Lindahl R Hempel J. Wang B.C. Nat. Struct. Biol. 1997; 4: 317-326Crossref PubMed Scopus (273) Google Scholar, 7Steinmetz C.G. Xie P. Weiner H. Hurley T.D. Structure. 1997; 5: 701-711Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar, 8Moore S.A. Baker H.M. Blythe T.J. Kitson K.E. Kitson T.M. Baker E.N. Structure. 1998; 6: 1541-1551Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, 9Lamb A.L. Newcomer M.E. Biochemistry. 1999; 38: 6003-6011Crossref PubMed Scopus (84) Google Scholar, 10Johansson K., El- Ahmad M. Ramaswamy S. Hjelmqvist L. Jornvall H. Eklund H. Protein Sci. 1998; 7: 2106-2117Crossref PubMed Scopus (133) Google Scholar, 11Ni L. Zhou J. Hurley T.D. Weiner H. Protein Sci. 1999; 8: 2784-2790Crossref PubMed Scopus (72) Google Scholar, 12Ahvazi B. Coulombe R. Delarge M. Vedadi M. Zhang L. Meighen E. Vrielink A. Biochem. J. 2000; 349: 853-861Crossref PubMed Scopus (73) Google Scholar, 13Pohl E. Brunner N. Wilmanns M. Hensel R. J. Biol. Chem. 2002; 277: 19938-19945Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar)) gives no clear information on the binding mode of the aldehyde substrate. Therefore, characterizing the molecular and structural factors involved in substrate specificity is important for a better understanding of the efficiency of the catalytic event and of the evolution of the active sites of ALDHs.Unlike most ALDHs, GAPN is an ALDH for which the physiological substrate is known. The fact that GAPN shows high catalytic efficiency toward d-G3P implies prerequisites with respect to the chemical mechanism. Previous enzymatic and structural studies of GAPN from Streptococcus mutans have shown that NADP binding induces a local conformational rearrangement. As a consequence, the thiol of Cys-302 becomes accessible, its pK appshifts from 8.5 to 6.2, and it is now well positioned to subsequently form a competent thiohemiacetal intermediate. On the other hand, the side chains of Glu-268 and Arg-459 also rotate and then do not interact with each other anymore. The side chain of Arg-459 is now orientated toward the substrate binding site. Glu-268 now has a pK app that shifts from probably 5.7 in the apo structure to 7.6 in the holo form and has its side chain well positioned for subsequently activating the water molecule involved in hydrolysis of the thioacyl intermediate. During this rearrangement, the distance between Glu-268 and Cys-302 passes from 7.6 to 3.6 Å. Concomitantly, the oxyanion hole, composed of at least the amide side chain of Asn-169 and the NH main chain of Cys-302, is formed. Its role is to stabilize the tetrahedral transition states formed during acylation and deacylation steps. This local conformational change has been shown to be strongly favored by binding of d-G3P to the binary complex GAPN·NADP (14Marchal S. Branlant G. Biochemistry. 1999; 38: 12950-15958Crossref PubMed Scopus (64) Google Scholar, 15Cobessi D. Tête-Favier F. Marchal S. Branlant G. Aubry A. J. Mol. Biol. 2000; 300: 141-152Crossref PubMed Scopus (81) Google Scholar, 16Marchal S. Rahuel-Clermont S. Branlant G. Biochemistry. 2000; 39: 3327-3335Crossref PubMed Scopus (49) Google Scholar). Whereas the rate-limiting step of GAPN is deacylation, that of human liver mitochondrial ALDH has been shown to depend on the chemical structure of the aldehyde substrate (17Ni L. Sheikh S. Weiner H. J. Biol. Chem. 1997; 272: 18823-188236Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Thus, it appears that the nature of the substrate can modulate the formation of a competent ternary complex.This justifies the determination of the structural factors that are implied in the catalytic efficiency of GAPNs. Several crystal structures of GAPN from S. mutans have been described so far (15Cobessi D. Tête-Favier F. Marchal S. Branlant G. Aubry A. J. Mol. Biol. 2000; 300: 141-152Crossref PubMed Scopus (81) Google Scholar, 18Cobessi D. Tête-Favier F. Marchal S. Azza S. Branlant G. Aubry A. J. Mol. Biol. 1999; 290: 161-173Crossref PubMed Scopus (87) Google Scholar). The apo1 structure shows a sulfate anion, called SO4a, which probably corresponds to the C-3 phosphate of G3P. Arg-124 and Arg-301, which are located in an α-helix and within a loop, respectively, interact with SO4a via their guanidinium groups. Arg-459, which is located within a loop, also interacts with SO4a but via its NH main chain. The apo2 structure shows a second sulfate anion, called SO4b, which probably mimics the tetrahedral transition states involved in acylation and deacylation. Guanidinium groups of Arg-301 and Arg-459 interact with SO4b. SO4b is also in interaction with the Asn-169 amide group, the Cys-302 and Thr-303 main chain NH, and the Thr-303 hydroxyl group, which in turn also interacts with SO4a. Inspection of the structure of the ternary complex C302S-NADP-G3P supports the potential roles of Arg-124, Arg-301, Arg-459, and Thr-303 in d-G3P oxidation process but also of the hydroxyl of Tyr-170, which forms a hydrogen bond with the oxygen that bridges the carbon chain to the phosphate in d-G3P. On the basis of sequence comparison of GAPNs to those of other ALDHs (Fig.1) only Arg-124, Arg-301, Arg-459, and Tyr-170 seem to be specific to GAPNs. Here, Arg-124 and Arg-301 were substituted by leucine, and Arg-459 and Tyr-170 were changed into isoleucine and phenylalanine, respectively. The catalytic properties of these GAPN mutants were determined. Altogether, the results suggest that Tyr-170 has no significant role, Arg-124 participates only ind-G3P binding via interaction with C-3 phosphate, and both Arg-301 and Arg-459 are not only involved in d-G3P binding but also participate in positioning efficiently the substrate with respect to Cys-302 within the ternary complex GAPN·NADP·d-G3P.DISCUSSIONGAPN belongs to the ALDH family. It is one of the ALDHs whose properties have been the most studied at both the structural and enzymatic levels. The fact that all of these studies were carried out with the physiological substrate d-G3P is also a guarantee of the generality of the interpretations of the results. Taking into account all of the enzymatic and structural data available so far, a scenario of the catalytic mechanism of GAPN from S. mutanscan be described as presented in the Introduction. As shown, the binding of NADP to apo-GAPN induces a local conformational change of the active site with at least a reorientation of side chains of Cys-302, Glu-268, and Arg-459. The rate of the conformational change upon cofactor binding has a maximal k obs value of 4.7 × 10−4 s−1 at acidic pH, which is not compatible with the k ac andk cat values (14Marchal S. Branlant G. Biochemistry. 1999; 38: 12950-15958Crossref PubMed Scopus (64) Google Scholar). The fact that addingd-G3P increases the rate of the reorganization by a factor of at least 105-fold (14Marchal S. Branlant G. Biochemistry. 1999; 38: 12950-15958Crossref PubMed Scopus (64) Google Scholar) 6S. Marchal and G. Branlant, unpublished results. indicates thatd-G3P binding to the binary complex GAPN·NADP strongly favors the active site reorganization. Therefore, characterizing the structural factors involved in d-G3P binding could help to a better understanding of the catalysis and of the evolution of the substrate binding sites of ALDHs. As presented in the Introduction, four residues seem to be conserved in the GAPN family (i.e.Arg-124, Tyr-170, Arg-301, and Arg-459) and could be involved in the recognition of d-G3P and/or in catalysis.Substituting Arg-124 with leucine does not change the nature of 1) the rate-limiting step, which remains deacylation, and 2) the rate-determining step in acylation, which remains hydride transfer. Moreover, no significant effect is observed on acylation and deacylation rates. Therefore, these results exclude a role of the guanidinium group of Arg-124 in stabilizing the transition states associated with hydride transfer and involved in hydrolysis. Only an increase in K m is observed in both acylation and deacylation steps but with a more pronounced effect in the latter step. Therefore, the only role of Arg-124 is to stabilize the binding of G3P via an interaction between the guanidinium group and the phosphate at C-3. This is in accord with the structure of 1) the ternary complex C302S·NADP·d-G3P in which the guanidinium group of Arg-124 interacts with one of the oxygens of the phosphate and 2) the apo2 form in which the guanidinium group interacts with one of the oxygens of the anion SO4a that is postulated to mimic the phosphate of d-G3P. The fact that the pK app of Cys-302 within the acylation complex remains similar to that observed in wild type indicates no role of Arg-124 in activating Cys-302. Again, this is in accord with the x-ray structure of apo2 GAPN, which shows a distance between both residues higher than 12 Å.The situation is different for the Y170F mutant GAPN protein. No major effect is observed in the K m values ford-G3P and in the rates of both acylation and deacylation steps. Therefore, although the hydroxyl group of Tyr-170 within the ternary complex C302S·NADP·d-G3P has been shown to be at a hydrogen bonding distance of the oxygen that connects the C-3 carbon to the phosphate in d-G3P, the interaction seems not to be essential for d-G3P binding. The fact that, in some archeal GAPNs, Tyr-170 is replaced by a phenylalanine supports the present results (Fig. 1).The behavior of the R459I mutant GAPN protein is very different. With this, the acylation process becomes rate-limiting with a limiting step prior to hydride transfer. The efficiency of the acylation step is strongly decreased by at least a factor of 105, which includes both a K m and ak ac contributions. From different approaches,i.e. using kinetic probes like 2PDS and the Ab333 or tracing the pH rate profile of acylation and of Ab333 appearance, it has been possible to determine the pK app of Cys-302. Whatever the approaches, a pK app of 6.6–6.7 is found within either the holo form or the ternary complex. Therefore, the guanidinium group of Arg-459, which is situated 5.7 Å from Cys-302 in the apo2 structure appears to participate directly or indirectly in lowering the pK app of Cys-302 by 0.5 pH units. However, the 0.5-unit increase in pK app could not explain the drastic decrease in k ac. In fact, as shown by using 2PDS probe, Cys-302 is at least 2 × 103-fold less reactive within the R459I holo form than within the holo wild type. Thus, Cys-302 has little accessibility in the R459I holo form. Therefore, it is probable that the location of the side chain of Cys-302 is not optimal and then necessitates a local reorganization for forming a competent ternary complex with d-G3P that could be rate-limiting. Several data favor this assumption. First, the Ab333 is not observed in holo wild type. This supports a relative positioning of Cys-302 and of the nicotinamide ring of NADP in R459I mutant different from that of wild type. Second, pH dependence of Ab333 of R459I mutant depends on Cys-302 pK app but not on Glu-268 pK app. This is an indication of a relative positioning of Cys-302 and Glu-268 in the R459I holo form different from that observed in competent holo wild type in which the distance between the side chains of Cys-302 and Glu-268 is around 3.6 Å. As seen from the inspection of the apo2 structure of GAPN, Arg-459 main chain NH interacts with the SO4a, which is representative of the phosphate of d-G3P, whereas guanidinium side chain interacts with SO4b, which probably mimics the sp3 transition states. Arg-459 interacts also with Gln-455, which is a residue conserved in almost all GAPNs. Therefore, substituting Arg-459 by isoleucine can also modify the conformation of the loop on which Arg-459 is located. Together, this is in accord with the data of the present study, which shows that substituting Arg-459 by isoleucine has not only a destabilizing K m effect for d-G3P but also a strong kinetic effect, which reflects a Michaelis ternary complex that seems to be inefficient for forming the thiohemiacetal intermediate ternary complex.The role of Arg-301 is more difficult to comprehend in detail due the very low catalytic efficiency of R301L mutant GAPN protein. As with the R459I mutant, the rate-limiting step is associated with acylation, more precisely prior to hydride transfer, and a high K mvalue for d-G3P and a low k ac value are observed. Taking into account the apo2 structure of GAPN, which shows an interaction of Arg-301 guanidinium side chain with both the SO4a and SO4b, it is probable that substituting Arg-301 by leucine should destabilize d-G3P binding but at the same time perturb the relative positioning of d-G3P and of Cys-302 within the ternary complex. Consequently, this would prevent an efficient attack of the thiolate group toward the aldehydic group.GAPN from S. mutans reduces efficiently d andl isomers of G3P (16Marchal S. Rahuel-Clermont S. Branlant G. Biochemistry. 2000; 39: 3327-3335Crossref PubMed Scopus (49) Google Scholar). Therefore, Arg-301 and Arg-459 are probably not involved in G3P stereospecificity. This suggests that both arginines do not interact selectively with the C-2 moiety ofd-G3P. This is also supported by the observation that both residues are conserved in GAPNs from plants which are described to reduce only d isomer (22Gomez Casati D.F. Sesma J.I. Iglesias A.A. Plant Sci. 2000; 154: 107-115Crossref PubMed Scopus (28) Google Scholar). Thus, as supported by the present study, one major role of Arg-301 and Arg-459 would be to position efficiently the d-G3P with respect to Cys-302 within the Michaelis ternary complex. This would favor the formation of the thiohemiacetal intermediate and consequently the efficiency of the hydride transfer. During this part of the catalytic event, the geometry at the C-1 carbon changes from sp2 to sp3 and reveals the role of Asn-169. Now, the negative charge of the oxygen of the thiohemiacetal intermediate is stabilized by the oxyanion site, which is composed at least of the amide side chain of Asn-169. This is in accord with previous studies, which showed that mutating Asn-169 results in a drastic decrease of the hydride transfer rate but has no effect on K m value for d-G3P (15Cobessi D. Tête-Favier F. Marchal S. Branlant G. Aubry A. J. Mol. Biol. 2000; 300: 141-152Crossref PubMed Scopus (81) Google Scholar). Nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN)1 catalyzes the irreversible oxidation of d-glyceraldehyde 3-phosphate (d-G3P) into 3-phosphoglycerate in the presence of NADP via a two-step chemical mechanism. It belongs to the aldehyde dehydrogenase (ALDH) superfamily, which oxidizes a wide variety of aldehydes into nonactivated or SCoA-activated acidic compounds (1Lindahl R. Crit. Rev. Biochem. Mol. Biol. 1992; 27: 283-335Crossref PubMed Scopus (360) Google Scholar, 2Vasiliou V. Pappa A. Petersen D.R. Chem. Biol. Interact. 2000; 129: 1-19Crossref PubMed Scopus (298) Google Scholar, 3Vasiliou V. Pappa A. Pharmacology. 2000; 61: 192-198Crossref PubMed Scopus (134) Google Scholar). These enzymes play parts at several levels of cellular metabolism including anabolic and catabolic pathways, detoxification processes, and embryogenesis development. Their numerous functions probably explain why most ALDHs show a wide substrate specificity, except for the CoA-dependent ALDH. In mammals, ALDH class 1 shows preference for retinal but it can also oxidize aromatic and “long-chain” aldehydes with dissimilar catalytic efficiencies, whereas ALDHs that belong to class 2 are more specific for acetaldehyde and short-chain aldehydes (4Klyosov A.A. Rashkovetsky L.G. Tahir M.K. Keung W.M. Biochemistry. 1996; 35: 4445-4456Crossref PubMed Scopus (155) Google Scholar, 5Klyosov A.A. Biochemistry. 1996; 35: 4457-4467Crossref PubMed Scopus (126) Google Scholar). Inspection of the active site of the ALDH structures (i.e. rat dimeric class 3 ALDH, bovine mitochondrial ALDH, sheep liver cytosolic ALDH, rat retinal dehydrogenase type II, cod liver betaine ALDH, human liver mitochondrial ALDH, ALDH from Vibrio harveyi, and GAPN fromThermoproteus tenax (6Liu Z.J. Sun Y.J. Rose J. Chung Y.J. Hsiao C.D. Chang W.R. Kuo I. Perozich J. Lindahl R Hempel J. Wang B.C. Nat. Struct. Biol. 1997; 4: 317-326Crossref PubMed Scopus (273) Google Scholar, 7Steinmetz C.G. Xie P. Weiner H. Hurley T.D. Structure. 1997; 5: 701-711Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar, 8Moore S.A. Baker H.M. Blythe T.J. Kitson K.E. Kitson T.M. Baker E.N. Structure. 1998; 6: 1541-1551Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, 9Lamb A.L. Newcomer M.E. Biochemistry. 1999; 38: 6003-6011Crossref PubMed Scopus (84) Google Scholar, 10Johansson K., El- Ahmad M. Ramaswamy S. Hjelmqvist L. Jornvall H. Eklund H. Protein Sci. 1998; 7: 2106-2117Crossref PubMed Scopus (133) Google Scholar, 11Ni L. Zhou J. Hurley T.D. Weiner H. Protein Sci. 1999; 8: 2784-2790Crossref PubMed Scopus (72) Google Scholar, 12Ahvazi B. Coulombe R. Delarge M. Vedadi M. Zhang L. Meighen E. Vrielink A. Biochem. J. 2000; 349: 853-861Crossref PubMed Scopus (73) Google Scholar, 13Pohl E. Brunner N. Wilmanns M. Hensel R. J. Biol. Chem. 2002; 277: 19938-19945Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar)) gives no clear information on the binding mode of the aldehyde substrate. Therefore, characterizing the molecular and structural factors involved in substrate specificity is important for a better understanding of the efficiency of the catalytic event and of the evolution of the active sites of ALDHs. Unlike most ALDHs, GAPN is an ALDH for which the physiological substrate is known. The fact that GAPN shows high catalytic efficiency toward d-G3P implies prerequisites with respect to the chemical mechanism. Previous enzymatic and structural studies of GAPN from Streptococcus mutans have shown that NADP binding induces a local conformational rearrangement. As a consequence, the thiol of Cys-302 becomes accessible, its pK appshifts from 8.5 to 6.2, and it is now well positioned to subsequently form a competent thiohemiacetal intermediate. On the other hand, the side chains of Glu-268 and Arg-459 also rotate and then do not interact with each other anymore. The side chain of Arg-459 is now orientated toward the substrate binding site. Glu-268 now has a pK app that shifts from probably 5.7 in the apo structure to 7.6 in the holo form and has its side chain well positioned for subsequently activating the water molecule involved in hydrolysis of the thioacyl intermediate. During this rearrangement, the distance between Glu-268 and Cys-302 passes from 7.6 to 3.6 Å. Concomitantly, the oxyanion hole, composed of at least the amide side chain of Asn-169 and the NH main chain of Cys-302, is formed. Its role is to stabilize the tetrahedral transition states formed during acylation and deacylation steps. This local conformational change has been shown to be strongly favored by binding of d-G3P to the binary complex GAPN·NADP (14Marchal S. Branlant G. Biochemistry. 1999; 38: 12950-15958Crossref PubMed Scopus (64) Google Scholar, 15Cobessi D. Tête-Favier F. Marchal S. Branlant G. Aubry A. J. Mol. Biol. 2000; 300: 141-152Crossref PubMed Scopus (81) Google Scholar, 16Marchal S. Rahuel-Clermont S. Branlant G. Biochemistry. 2000; 39: 3327-3335Crossref PubMed Scopus (49) Google Scholar). Whereas the rate-limiting step of GAPN is deacylation, that of human liver mitochondrial ALDH has been shown to depend on the chemical structure of the aldehyde substrate (17Ni L. Sheikh S. Weiner H. J. Biol. Chem. 1997; 272: 18823-188236Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Thus, it appears that the nature of the substrate can modulate the formation of a competent ternary complex. This justifies the determination of the structural factors that are implied in the catalytic efficiency of GAPNs. Several crystal structures of GAPN from S. mutans have been described so far (15Cobessi D. Tête-Favier F. Marchal S. Branlant G. Aubry A. J. Mol. Biol. 2000; 300: 141-152Crossref PubMed Scopus (81) Google Scholar, 18Cobessi D. Tête-Favier F. Marchal S. Azza S. Branlant G. Aubry A. J. Mol. Biol. 1999; 290: 161-173Crossref PubMed Scopus (87) Google Scholar). The apo1 structure shows a sulfate anion, called SO4a, which probably corresponds to the C-3 phosphate of G3P. Arg-124 and Arg-301, which are located in an α-helix and within a loop, respectively, interact with SO4a via their guanidinium groups. Arg-459, which is located within a loop, also interacts with SO4a but via its NH main chain. The apo2 structure shows a second sulfate anion, called SO4b, which probably mimics the tetrahedral transition states involved in acylation and deacylation. Guanidinium groups of Arg-301 and Arg-459 interact with SO4b. SO4b is also in interaction with the Asn-169 amide group, the Cys-302 and Thr-303 main chain NH, and the Thr-303 hydroxyl group, which in turn also interacts with SO4a. Inspection of the structure of the ternary complex C302S-NADP-G3P supports the potential roles of Arg-124, Arg-301, Arg-459, and Thr-303 in d-G3P oxidation process but also of the hydroxyl of Tyr-170, which forms a hydrogen bond with the oxygen that bridges the carbon chain to the phosphate in d-G3P. On the basis of sequence comparison of GAPNs to those of other ALDHs (Fig.1) only Arg-124, Arg-301, Arg-459, and Tyr-170 seem to be specific to GAPNs. Here, Arg-124 and Arg-301 were substituted by leucine, and Arg-459 and Tyr-170 were changed into isoleucine and phenylalanine, respectively. The catalytic properties of these GAPN mutants were determined. Altogether, the results suggest that Tyr-170 has no significant role, Arg-124 participates only ind-G3P binding via interaction with C-3 phosphate, and both Arg-301 and Arg-459 are not only involved in d-G3P binding but also participate in positioning efficiently the substrate with respect to Cys-302 within the ternary complex GAPN·NADP·d-G3P. DISCUSSIONGAPN belongs to the ALDH family. It is one of the ALDHs whose properties have been the most studied at both the structural and enzymatic levels. The fact that all of these studies were carried out with the physiological substrate d-G3P is also a guarantee of the generality of the interpretations of the results. Taking into account all of the enzymatic and structural data available so far, a scenario of the catalytic mechanism of GAPN from S. mutanscan be described as presented in the Introduction. As shown, the binding of NADP to apo-GAPN induces a local conformational change of the active site with at least a reorientation of side chains of Cys-302, Glu-268, and Arg-459. The rate of the conformational change upon cofactor binding has a maximal k obs value of 4.7 × 10−4 s−1 at acidic pH, which is not compatible with the k ac andk cat values (14Marchal S. Branlant G. Biochemistry. 1999; 38: 12950-15958Crossref PubMed Scopus (64) Google Scholar). The fact that addingd-G3P increases the rate of the reorganization by a factor of at least 105-fold (14Marchal S. Branlant G. Biochemistry. 1999; 38: 12950-15958Crossref PubMed Scopus (64) Google Scholar) 6S. Marchal and G. Branlant, unpublished results. indicates thatd-G3P binding to the binary complex GAPN·NADP strongly favors the active site reorganization. Therefore, characterizing the structural factors involved in d-G3P binding could help to a better understanding of the catalysis and of the evolution of the substrate binding sites of ALDHs. As presented in the Introduction, four residues seem to be conserved in the GAPN family (i.e.Arg-124, Tyr-170, Arg-301, and Arg-459) and could be involved in the recognition of d-G3P and/or in catalysis.Substituting Arg-124 with leucine does not change the nature of 1) the rate-limiting step, which remains deacylation, and 2) the rate-determining step in acylation, which remains hydride transfer. Moreover, no significant effect is observed on acylation and deacylation rates. Therefore, these results exclude a role of the guanidinium group of Arg-124 in stabilizing the transition states associated with hydride transfer and involved in hydrolysis. Only an increase in K m is observed in both acylation and deacylation steps but with a more pronounced effect in the latter step. Therefore, the only role of Arg-124 is to stabilize the binding of G3P via an interaction between the guanidinium group and the phosphate at C-3. This is in accord with the structure of 1) the ternary complex C302S·NADP·d-G3P in which the guanidinium group of Arg-124 interacts with one of the oxygens of the phosphate and 2) the apo2 form in which the guanidinium group interacts with one of the oxygens of the anion SO4a that is postulated to mimic the phosphate of d-G3P. The fact that the pK app of Cys-302 within the acylation complex remains similar to that observed in wild type indicates no role of Arg-124 in activating Cys-302. Again, this is in accord with the x-ray structure of apo2 GAPN, which shows a distance between both residues higher than 12 Å.The situation is different for the Y170F mutant GAPN protein. No major effect is observed in the K m values ford-G3P and in the rates of both acylation and deacylation steps. Therefore, although the hydroxyl group of Tyr-170 within the ternary complex C302S·NADP·d-G3P has been shown to be at a hydrogen bonding distance of the oxygen that connects the C-3 carbon to the phosphate in d-G3P, the interaction seems not to be essential for d-G3P binding. The fact that, in some archeal GAPNs, Tyr-170 is replaced by a phenylalanine supports the present results (Fig. 1).The behavior of the R459I mutant GAPN protein is very different. With this, the acylation process becomes rate-limiting with a limiting step prior to hydride transfer. The efficiency of the acylation step is strongly decreased by at least a factor of 105, which includes both a K m and ak ac contributions. From different approaches,i.e. using kinetic probes like 2PDS and the Ab333 or tracing the pH rate profile of acylation and of Ab333 appearance, it has been possible to determine the pK app of Cys-302. Whatever the approaches, a pK app of 6.6–6.7 is found within either the holo form or the ternary complex. Therefore, the guanidinium group of Arg-459, which is situated 5.7 Å from Cys-302 in the apo2 structure appears to participate directly or indirectly in lowering the pK app of Cys-302 by 0.5 pH units. However, the 0.5-unit increase in pK app could not explain the drastic decrease in k ac. In fact, as shown by using 2PDS probe, Cys-302 is at least 2 × 103-fold less reactive within the R459I holo form than within the holo wild type. Thus, Cys-302 has little accessibility in the R459I holo form. Therefore, it is probable that the location of the side chain of Cys-302 is not optimal and then necessitates a local reorganization for forming a competent ternary complex with d-G3P that could be rate-limiting. Several data favor this assumption. First, the Ab333 is not observed in holo wild type. This supports a relative positioning of Cys-302 and of the nicotinamide ring of NADP in R459I mutant different from that of wild type. Second, pH dependence of Ab333 of R459I mutant depends on Cys-302 pK app but not on Glu-268 pK app. This is an indication of a relative positioning of Cys-302 and Glu-268 in the R459I holo form different from that observed in competent holo wild type in which the distance between the side chains of Cys-302 and Glu-268 is around 3.6 Å. As seen from the inspection of the apo2 structure of GAPN, Arg-459 main chain NH interacts with the SO4a, which is representative of the phosphate of d-G3P, whereas guanidinium side chain interacts with SO4b, which probably mimics the sp3 transition states. Arg-459 interacts also with Gln-455, which is a residue conserved in almost all GAPNs. Therefore, substituting Arg-459 by isoleucine can also modify the conformation of the loop on which Arg-459 is located. Together, this is in accord with the data of the present study, which shows that substituting Arg-459 by isoleucine has not only a destabilizing K m effect for d-G3P but also a strong kinetic effect, which reflects a Michaelis ternary complex that seems to be inefficient for forming the thiohemiacetal intermediate ternary complex.The role of Arg-301 is more difficult to comprehend in detail due the very low catalytic efficiency of R301L mutant GAPN protein. As with the R459I mutant, the rate-limiting step is associated with acylation, more precisely prior to hydride transfer, and a high K mvalue for d-G3P and a low k ac value are observed. Taking into account the apo2 structure of GAPN, which shows an interaction of Arg-301 guanidinium side chain with both the SO4a and SO4b, it is probable that substituting Arg-301 by leucine should destabilize d-G3P binding but at the same time perturb the relative positioning of d-G3P and of Cys-302 within the ternary complex. Consequently, this would prevent an efficient attack of the thiolate group toward the aldehydic group.GAPN from S. mutans reduces efficiently d andl isomers of G3P (16Marchal S. Rahuel-Clermont S. Branlant G. Biochemistry. 2000; 39: 3327-3335Crossref PubMed Scopus (49) Google Scholar). Therefore, Arg-301 and Arg-459 are probably not involved in G3P stereospecificity. This suggests that both arginines do not interact selectively with the C-2 moiety ofd-G3P. This is also supported by the observation that both residues are conserved in GAPNs from plants which are described to reduce only d isomer (22Gomez Casati D.F. Sesma J.I. Iglesias A.A. Plant Sci. 2000; 154: 107-115Crossref PubMed Scopus (28) Google Scholar). Thus, as supported by the present study, one major role of Arg-301 and Arg-459 would be to position efficiently the d-G3P with respect to Cys-302 within the Michaelis ternary complex. This would favor the formation of the thiohemiacetal intermediate and consequently the efficiency of the hydride transfer. During this part of the catalytic event, the geometry at the C-1 carbon changes from sp2 to sp3 and reveals the role of Asn-169. Now, the negative charge of the oxygen of the thiohemiacetal intermediate is stabilized by the oxyanion site, which is composed at least of the amide side chain of Asn-169. This is in accord with previous studies, which showed that mutating Asn-169 results in a drastic decrease of the hydride transfer rate but has no effect on K m value for d-G3P (15Cobessi D. Tête-Favier F. Marchal S. Branlant G. Aubry A. J. Mol. Biol. 2000; 300: 141-152Crossref PubMed Scopus (81) Google Scholar). GAPN belongs to the ALDH family. It is one of the ALDHs whose properties have been the most studied at both the structural and enzymatic levels. The fact that all of these studies were carried out with the physiological substrate d-G3P is also a guarantee of the generality of the interpretations of the results. Taking into account all of the enzymatic and structural data available so far, a scenario of the catalytic mechanism of GAPN from S. mutanscan be described as presented in the Introduction. As shown, the binding of NADP to apo-GAPN induces a local conformational change of the active site with at least a reorientation of side chains of Cys-302, Glu-268, and Arg-459. The rate of the conformational change upon cofactor binding has a maximal k obs value of 4.7 × 10−4 s−1 at acidic pH, which is not compatible with the k ac andk cat values (14Marchal S. Branlant G. Biochemistry. 1999; 38: 12950-15958Crossref PubMed Scopus (64) Google Scholar). The fact that addingd-G3P increases the rate of the reorganization by a factor of at least 105-fold (14Marchal S. Branlant G. Biochemistry. 1999; 38: 12950-15958Crossref PubMed Scopus (64) Google Scholar) 6S. Marchal and G. Branlant, unpublished results. indicates thatd-G3P binding to the binary complex GAPN·NADP strongly favors the active site reorganization. Therefore, characterizing the structural factors involved in d-G3P binding could help to a better understanding of the catalysis and of the evolution of the substrate binding sites of ALDHs. As presented in the Introduction, four residues seem to be conserved in the GAPN family (i.e.Arg-124, Tyr-170, Arg-301, and Arg-459) and could be involved in the recognition of d-G3P and/or in catalysis. Substituting Arg-124 with leucine does not change the nature of 1) the rate-limiting step, which remains deacylation, and 2) the rate-determining step in acylation, which remains hydride transfer. Moreover, no significant effect is observed on acylation and deacylation rates. Therefore, these results exclude a role of the guanidinium group of Arg-124 in stabilizing the transition states associated with hydride transfer and involved in hydrolysis. Only an increase in K m is observed in both acylation and deacylation steps but with a more pronounced effect in the latter step. Therefore, the only role of Arg-124 is to stabilize the binding of G3P via an interaction between the guanidinium group and the phosphate at C-3. This is in accord with the structure of 1) the ternary complex C302S·NADP·d-G3P in which the guanidinium group of Arg-124 interacts with one of the oxygens of the phosphate and 2) the apo2 form in which the guanidinium group interacts with one of the oxygens of the anion SO4a that is postulated to mimic the phosphate of d-G3P. The fact that the pK app of Cys-302 within the acylation complex remains similar to that observed in wild type indicates no role of Arg-124 in activating Cys-302. Again, this is in accord with the x-ray structure of apo2 GAPN, which shows a distance between both residues higher than 12 Å. The situation is different for the Y170F mutant GAPN protein. No major effect is observed in the K m values ford-G3P and in the rates of both acylation and deacylation steps. Therefore, although the hydroxyl group of Tyr-170 within the ternary complex C302S·NADP·d-G3P has been shown to be at a hydrogen bonding distance of the oxygen that connects the C-3 carbon to the phosphate in d-G3P, the interaction seems not to be essential for d-G3P binding. The fact that, in some archeal GAPNs, Tyr-170 is replaced by a phenylalanine supports the present results (Fig. 1). The behavior of the R459I mutant GAPN protein is very different. With this, the acylation process becomes rate-limiting with a limiting step prior to hydride transfer. The efficiency of the acylation step is strongly decreased by at least a factor of 105, which includes both a K m and ak ac contributions. From different approaches,i.e. using kinetic probes like 2PDS and the Ab333 or tracing the pH rate profile of acylation and of Ab333 appearance, it has been possible to determine the pK app of Cys-302. Whatever the approaches, a pK app of 6.6–6.7 is found within either the holo form or the ternary complex. Therefore, the guanidinium group of Arg-459, which is situated 5.7 Å from Cys-302 in the apo2 structure appears to participate directly or indirectly in lowering the pK app of Cys-302 by 0.5 pH units. However, the 0.5-unit increase in pK app could not explain the drastic decrease in k ac. In fact, as shown by using 2PDS probe, Cys-302 is at least 2 × 103-fold less reactive within the R459I holo form than within the holo wild type. Thus, Cys-302 has little accessibility in the R459I holo form. Therefore, it is probable that the location of the side chain of Cys-302 is not optimal and then necessitates a local reorganization for forming a competent ternary complex with d-G3P that could be rate-limiting. Several data favor this assumption. First, the Ab333 is not observed in holo wild type. This supports a relative positioning of Cys-302 and of the nicotinamide ring of NADP in R459I mutant different from that of wild type. Second, pH dependence of Ab333 of R459I mutant depends on Cys-302 pK app but not on Glu-268 pK app. This is an indication of a relative positioning of Cys-302 and Glu-268 in the R459I holo form different from that observed in competent holo wild type in which the distance between the side chains of Cys-302 and Glu-268 is around 3.6 Å. As seen from the inspection of the apo2 structure of GAPN, Arg-459 main chain NH interacts with the SO4a, which is representative of the phosphate of d-G3P, whereas guanidinium side chain interacts with SO4b, which probably mimics the sp3 transition states. Arg-459 interacts also with Gln-455, which is a residue conserved in almost all GAPNs. Therefore, substituting Arg-459 by isoleucine can also modify the conformation of the loop on which Arg-459 is located. Together, this is in accord with the data of the present study, which shows that substituting Arg-459 by isoleucine has not only a destabilizing K m effect for d-G3P but also a strong kinetic effect, which reflects a Michaelis ternary complex that seems to be inefficient for forming the thiohemiacetal intermediate ternary complex. The role of Arg-301 is more difficult to comprehend in detail due the very low catalytic efficiency of R301L mutant GAPN protein. As with the R459I mutant, the rate-limiting step is associated with acylation, more precisely prior to hydride transfer, and a high K mvalue for d-G3P and a low k ac value are observed. Taking into account the apo2 structure of GAPN, which shows an interaction of Arg-301 guanidinium side chain with both the SO4a and SO4b, it is probable that substituting Arg-301 by leucine should destabilize d-G3P binding but at the same time perturb the relative positioning of d-G3P and of Cys-302 within the ternary complex. Consequently, this would prevent an efficient attack of the thiolate group toward the aldehydic group. GAPN from S. mutans reduces efficiently d andl isomers of G3P (16Marchal S. Rahuel-Clermont S. Branlant G. Biochemistry. 2000; 39: 3327-3335Crossref PubMed Scopus (49) Google Scholar). Therefore, Arg-301 and Arg-459 are probably not involved in G3P stereospecificity. This suggests that both arginines do not interact selectively with the C-2 moiety ofd-G3P. This is also supported by the observation that both residues are conserved in GAPNs from plants which are described to reduce only d isomer (22Gomez Casati D.F. Sesma J.I. Iglesias A.A. Plant Sci. 2000; 154: 107-115Crossref PubMed Scopus (28) Google Scholar). Thus, as supported by the present study, one major role of Arg-301 and Arg-459 would be to position efficiently the d-G3P with respect to Cys-302 within the Michaelis ternary complex. This would favor the formation of the thiohemiacetal intermediate and consequently the efficiency of the hydride transfer. During this part of the catalytic event, the geometry at the C-1 carbon changes from sp2 to sp3 and reveals the role of Asn-169. Now, the negative charge of the oxygen of the thiohemiacetal intermediate is stabilized by the oxyanion site, which is composed at least of the amide side chain of Asn-169. This is in accord with previous studies, which showed that mutating Asn-169 results in a drastic decrease of the hydride transfer rate but has no effect on K m value for d-G3P (15Cobessi D. Tête-Favier F. Marchal S. Branlant G. Aubry A. J. Mol. Biol. 2000; 300: 141-152Crossref PubMed Scopus (81) Google Scholar). We are indebted to Dr. F. Talfournier for participating in the achievement of this work. We are grateful to Dr. S. Azza and S. Boutserin for efficient technical help. We thank Dr. S. Rahuel-Clermont for valuable discussions. We also thank Dr. T. Barman for correcting the manuscript." @default.
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W2084566211 title "Characterization of the Amino Acids Involved in Substrate Specificity of Nonphosphorylating Glyceraldehyde-3-Phosphate Dehydrogenase from Streptococcus mutans" @default.
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