Structural analysis of glyceraldehyde 3-phosphate dehydrogenase from Escherichia coli: direct evidence of substrate binding and cofactor-induced conformational changes

The crystal structures of gyceraldehyde 3-phosphate dehydrogenase (GAPDH) from Escherichia coli have been determined in three different enzymatic states, NAD(+)-free, NAD(+)-bound, and hemiacetal intermediate. The NAD(+)-free structure reported here has been determined from monoclinic and tetragonal crystal forms. The conformational changes in GAPDH induced by cofactor binding are limited to the residues that bind the adenine moiety of NAD(+). Glyceraldehyde 3-phosphate (GAP), the substrate of GAPDH, binds to the enzyme with its C3 phosphate in a hydrophilic pocket, called the "new P(i)" site, which is different from the originally proposed binding site for inorganic phosphate. This observed location of the C3 phosphate is consistent with the flip-flop model proposed for the enzyme mechanism [Skarzynski, T., Moody, P. C., and Wonacott, A. J. (1987) J. Mol. Biol. 193, 171-187]. Via incorporation of the new P(i) site in this model, it is now proposed that the C3 phosphate of GAP initially binds at the new P(i) site and then flips to the P(s) site before hydride transfer. A superposition of NAD(+)-bound and hemiacetal intermediate structures reveals an interaction between the hydroxyl oxygen at the hemiacetal C1 of GAP and the nicotinamide ring. This finding suggests that the cofactor NAD(+) may stabilize the transition state oxyanion of the hemiacetal intermediate in support of the flip-flop model for GAP binding.     

Thermodynamics of the folding of D-glyceraldehyde-3-phosphate dehydrogenase assisted by protein disulfide isomerase studied by microcalorimetry.

Thermodynamics of the refolding of denatured D-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) assisted by protein disulfide isomerase (PDI), a molecular chaperone, has been studied by isothermal microcalorimetry at different molar ratios of PDI/GAPDH and temperatures using two thermodynamic models proposed for chaperone-substrate binding and chaperone-assisted substrate folding, respectively. The binding of GAPDH folding intermediates to PDI is driven by a large favorable enthalpy decrease with a large unfavorable entropy reduction, and shows strong enthalpy-entropy compensation and weak temperature dependence of Gibbs free energy change. A large negative heat-capacity change of the binding, -156 kJ.mol(-1).K(-1), at all temperatures examined indicates that hydrophobic interaction is a major force for the binding. The binding stoichiometry shows one dimeric GAPDH intermediate per PDI monomer. The refolding of GAPDH assisted by PDI is a largely exothermic reaction at 15.0-25.0 degrees C. With increasing temperature from 15.0 to 37.0 degrees C, the PDI-assisted reactivation yield of denatured GAPDH upon dilution decreases. At 37.0 degrees C, the spontaneous reactivation, PDI-assisted reactivation and intrinsic molar enthalpy change during the PDI-assisted refolding of GAPDH are not detected.     

Crystal structure of two ternary complexes of phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus with NAD and D-glyceraldehyde 3-phosphate

The crystal structure of the phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Bacillus stearothermophilus was solved in complex with its cofactor, NAD, and its physiological substrate, D-glyceraldehyde 3-phosphate (D-G3P). To isolate a stable ternary complex, the nucleophilic residue of the active site, Cys(149), was substituted with alanine or serine. The C149A and C149S GAPDH ternary complexes were obtained by soaking the crystals of the corresponding binary complexes (enzyme.NAD) in a solution containing G3P. The structures of the two binary and the two ternary complexes are presented. The D-G3P adopts the same conformation in the two ternary complexes. It is bound in a non-covalent way, in the free aldehyde form, its C-3 phosphate group being positioned in the P(s) site and not in the P(i) site. Its C-1 carbonyl oxygen points toward the essential His(176), which supports the role proposed for this residue along the two steps of the catalytic pathway. Arguments are provided that the structures reported here are representative of a productive enzyme.NAD.D-G3P complex in the ground state (Michaelis complex).     

Crystal structure of rabbit phosphoglucose isomerase complexed with its substrate D-fructose 6-phosphate.

Phosphoglucose isomerase (PGI, EC 5.3.1.9) catalyzes the interconversion of D-glucose 6-phosphate (G6P) and D-fructose 6-phosphate (F6P) and plays important roles in glycolysis and gluconeogenesis. Biochemical characterization of the enzyme has led to a proposed multistep catalytic mechanism. First, the enzyme catalyzes ring opening to yield the open chain form of the substrate. Then isomerization proceeds via proton transfer between C2 and C1 of a cis-enediol(ate) intermediate to yield the open chain form of the product. Catalysis proceeds in both the G6P to F6P and F6P to G6P directions, so both G6P and F6P are substrates. X-ray crystal structure analysis of rabbit and bacterial PGI has previously identified the location of the enzyme active site, and a recent crystal structure of rabbit PGI identified Glu357 as a candidate functional group for transferring the proton. However, it was not clear which active site amino acid residues catalyze the ring opening step. In this paper, we report the X-ray crystal structure of rabbit PGI complexed with the cyclic form of its substrate, D-fructose 6-phosphate, at 2.1 A resolution. The location of the substrate relative to the side chains of His388 suggest that His388 promotes ring opening by protonating the ring oxygen. Glu216 helps to position His388, and a water molecule that is held in position by Lys518 and Thr214 accepts a proton from the hydroxyl group at C2. Comparison to a structure of rabbit PGI with 5PAA bound indicates that ring opening is followed by loss of the protonated water molecule and conformational changes in the substrate and the protein so that a helix containing amino acids 513-520 moves in toward the substrate to form additional hydrogen bonds with the substrate.     

Evidence for the two phosphate binding sites of an analogue of the thioacyl intermediate for the Trypanosoma cruzi glyceraldehyde-3-phosphate dehydrogenase-catalyzed reaction,from its crystal structure

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzes the reversible oxidative phosphorylation of d-glyceraldehyde 3-phosphate (GAP) into d-glycerate 1,3-bisphosphate (1,3-diPG) in the presence of NAD(+) and inorganic phosphate (P(i)). Within the active site, two anion-binding sites were ascribed to the binding of the C3 phosphate of GAP (P(s)) and to the binding of the attacking phosphate ion (P(i)). The role played by these two sites in the catalytic mechanism in connection with the functional role of coenzyme exchange (NADH-NAD(+) shuttle) has been investigated by several studies leading to the C3 phosphate flipping model proposed by Skarzynski et al. [Skarzynski, T., Moody, P. C., and Wonacott, A. J. (1987) J. Mol. Biol. 193, 171-187]. This model has not yet received direct confirmation. To gain further insight into the role of both sites, we synthesized irreversible inhibitors which form with the essential cysteine residue a thioacyl enzyme analogue of the catalytic intermediate. Here we report the refined glycosomal Trypanosoma cruzi GAPDH in complex with a covalently bound GAP analogue at an improved resolution of 2.0-2.5 A. For this holo-thioacyl enzyme complex, a flip-flop movement is clearly characterized, the change from the P(i) to the P(s) binding site being correlated with the coenzyme exchange step: the weaker interaction of the intermediate when bound at the P(s) site with the cofactor allows its release and also the binding of the inorganic phosphate for the next catalytic step. This result gives strong experimental support for the generally accepted flip-flop model of the catalytic mechanism in GAPDH.     

Crystal Structure of Glyceraldehyde-3-Phosphate Dehydrogenase 1 from Methicillin-Resistant Staphylococcus aureus MRSA252 Provides Novel Insights into Substrate Binding and Catalytic Mechanism

The dreaded pathogen Staphylococcus aureus is one of the causes of morbidity and mortality worldwide. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), one of the key glycolytic enzymes, is irreversibly oxidized under oxidative stress and is responsible for sustenance of the pathogen inside the host. With an aim to elucidate the catalytic mechanism and identification of intermediates involved, we describe in this study different crystal structures of GAPDH1 from methicillin-resistant S. aureus MRSA252 (SaGAPDH1) in apo and holo forms of wild type, thioacyl intermediate, and ternary complexes of active-site mutants with physiological substrate d-glyceraldehyde-3-phosphate (G3P) and coenzyme NAD⁺. A new phosphate recognition site, “new P_i” site, similar to that observed in GAPDH from Thermotoga maritima, is reported here, which is 3.40 Å away from the “classical P_i” site. Ternary complexes discussed are representatives of noncovalent Michaelis complexes in the ground state. d-G3P is bound to all the four subunits of C151S.NAD and C151G.NAD in more reactive hydrate (gem-di-ol) form. However, in C151S + H178N.NAD, the substrate is bound to two chains in aldehyde form and in gem-di-ol form to the other two. This work reports binding of d-G3P to the C151G mutant in an inverted manner for the very first time. The structure of the thiaocyl complex presented here is formed after the hydride transfer. The C3 phosphate of d-G3P is positioned at the “P_s” site in the ternary complexes but at the “new P_i” site in the thioacyl complex and C1-O1 bond points opposite to His178 disrupting the alignment between itself and NE2 of His178. A new conformation (Conformation I) of the 209-215 loop has also been identified, where the interaction between phosphate ion at the “new P_i” site and conserved Gly212 is lost. Altogether, inferences drawn from the kinetic analyses and crystal structures suggest the “flip-flop” model proposed for the enzyme mechanism.     

Direct observation of the enzyme-intermediate complex of 5-enolpyruvylshikimate-3-phosphate synthase by 13C NMR spectroscopy

The interaction of 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase, 4,5-dideoxyshikimate 3-phosphate (ddS3P), and [2-13C]-and [3-13C]phosphoenolpyruvate (PEP) has been examined by 13C NMR spectroscopy. Although no resonances due to a dead-end intermediate complex could be detected, an enzyme active site specific formation of pyruvate was observed. The interaction of EPSP synthase with shikimate 3-phosphate (S3P) and [2-13C]- or [3-13C]PEP has been examined by 13C NMR spectroscopy. With [2-13C]PEP, in addition to the resonances due to [2-13C]PEP and [8-13C]EPSP, new resonances appeared at 164.8, 110.9, and 107.2 ppm. The resonance at 164.8 ppm has been assigned to enzyme-bound EPSP. The resonance at 110.9 ppm has been assigned to C-8 of an enzyme-free tetrahedral intermediate of the sort originally proposed by Levin and Sprinson [Levin, J. G., & Sprinson, D. B. (1964) J. Biol. Chem. 239, 1142-1150] and recently independently observed by Anderson et al. [Anderson, K. S., Sikorski, J. A., Benesi, A. J., & Johnson, K. A. (1988) J. Am. Chem. Soc. 110, 6577-6579]. The resonance at 107.2 ppm has been assigned to an enzyme-bound intermediate whose structure is closely related to that of the tetrahedral intermediate. With [3-13C]PEP, new resonances appeared at 88.9, 26.2, 25.5, and 24.5 ppm. The resonance at 88.9 ppm has been assigned to enzyme-bound EPSP. The resonance at 26.2 ppm, which was found to correlate with 1.48 ppm by isotope-edited multiple quantum coherence 1H NMR spectroscopy, has been assigned to the methyl group 4-hydroxy-4-methylketoglutarate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Computational study of glyceraldehyde-3-phosphate dehydrogenase of Entamoeba histolytica: implications for structure-based drug design

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of the pathogenic protozoa Entamoeba histolytica (Eh) is a major glycolytic enzyme and an attractive drug target since this parasite lacks a functional citric acid cycle and is dependent solely on glycolysis for its energy requirements. The three-dimensional structure of dimeric EhGAPDH in complex with cofactor NAD(+) has been generated by homology modeling based on the crystal structure of human liver GAPDH. Our refined model indicates the presence of a parasite specific disulfide bond between two cysteine residues of adjacent monomers in the EhGAPDH dimer, which may be an important target for future drug design. Flexible docking with the substrate glyceraldehyde-3-phosphate (G3P) shows that Cys151, His178, Thr210, and Arg233 are important residues in G3P binding. The inorganic phosphate-binding site of EhGAPDH has been determined by docking study. The binding mode of a natural GAPDH inhibitor, chalepin to EhGAPDH has also been predicted. In search for a better inhibitor for EhGADPH, in silico modification of chalepin has been carried out to form an additional specific polar interaction with Asp194 of EhGAPDH whose equivalent is Leu195 in human GAPDH. In the absence of a crystal structure, our study provides an early insight into the structure of major drug target EhGAPDH, thus, facilitating the inhibitor design.     

Engineered glycolytic glyceraldehyde-3-phosphate dehydrogenase binds the anti conformation of NAD+ nicotinamide but does not experience A-specific hydride transfer

Glycolytic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a NAD-dependent oxidoreductase which catalyzes the oxidative phosphorylation of d-glyceraldehyde-3-phosphate (G3P) to form 1, 3-diphosphoglycerate. The currently accepted mechanism involves an oxidoreduction step followed by a phosphorylation. GAPDH is classified as a B-specific oxidoreductase. The inspection of several crystal structures of GAPDHs indicates that the efficient hydride transfer from the hemithioacetal intermediate to the C4 position of the pyridinium si face requires optimal nicotinamidium-protein contacts for a suitable pyridinium-ring orientation. In previous studies carried out on Escherichia coli GAPDH (C. Corbier, A. Mougin, Y. Mely, H. W. Adolph, M. Zeppezauer, D. Gerard, A. Wonacott, and G. Branlant, Biochimie 72, 545-554, 1990; J. Eyschen, C. Corbier, B. Vitoux, G. Branlant, and M. T. Cung, Protein Pept. Lett. 1, 19-24, 1994), the role of the invariant Asn 313 residue, as an anchor which favors the syn orientation of the nicotinamide ring, was examined. Here, we report further investigations on the molecular factors responsible for the cofactor stereospecificity. Two single [Gly317] and [Ala317] GAPDH mutants and one double [Thr313-Gly317] GAPDH mutant were constructed on the basis of a molecular modelling study from the crystal structure of holo GAPDH from E. coli (E. Duée, L. Olivier-Deyris, E. Fanchon, C. Corbier, G. Branlant, and O. Dideberg, J. Mol. Biol. 257, 814-838, 1996). The Kd constants of [Ala317], [Gly317], and [Thr313-Gly317] GAPDH mutants for NAD are 5, 13, and 300 times higher than that of wild-type GAPDH. Transferred nuclear Overhauser effect spectroscopy demonstrates that the wild-type syn orientation of bound nicotinamide remains unchanged in the [Gly317] and [Ala317] mutants, whereas a conformational equilibrium between the syn and anti forms occurs in the [Thr313-Gly317] double mutant with a preference for the anti conformer. Although the double mutant preferably binds the nicotinamide ring in an anti conformation, it still exhibits B hydride transfer stereospecificity. Yet, the catalytic efficiency is much less than that of the wild type. This indicates that the holo GAPDH mutant fraction with an anti orientation of bound NAD is not capable of forming the ternary complex with G3P which would be required for an efficient A-specific catalytic process. The reasons of this catalytic inefficiency are discussed in relation with the historical and functional models which were advanced to explain the stereospecificity of NAD(P)-dependent dehydrogenases.     

P.versicolor龙虾D-甘油醛-3-磷酸脱氢酶的X射线晶体学研究——分子置换法结构测定

由P.versicolor龙虾尾肌提取的HOIO-D-甘油醛-3-磷酸脱氢酶(GAPDH),已长出可供Χ射线衍射用的晶体。初步Χ射线晶体学研究确定:此酶晶体属於C2空间群,不对称单位内含有半个分子,分子坐落在二重轴上。以Homarus Amercanus龙虾GAPDH结构为模型结构,应用分子置换技术进行了低分辨率Χ射线结构分析,结果表明:分子内亚基排列具有222对称性,分子Q轴平行于晶体学二重轴b,分子P和R轴分别平行于晶体学a和c轴。按分子置换法推出的结构模型算得5A分辨率的晶体学R因子为0.46。并获得了一套5A。分辨率的电子密度图。此酶的几种同晶型晶体,特别是荧光NAD衍生物晶体的较高分辨率的结构分析工作正在进行中。     

An unexpected phosphate binding site in Glyceraldehyde 3-Phosphate Dehydrogenase: Crystal structures of apo, holo and ternary complex of Cryptosporidium parvum enzyme

Background  

The structure, function and reaction mechanism of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) have been extensively studied. Based on these studies, three anion binding sites have been identified, one 'Ps' site (for binding the C-3 phosphate of the substrate) and two sites, 'Pi' and 'new Pi', for inorganic phosphate. According to the original flip-flop model, the substrate phosphate group switches from the 'Pi' to the 'Ps' site during the multistep reaction. In light of the discovery of the 'new Pi' site, a modified flip-flop mechanism, in which the C-3 phosphate of the substrate binds to the 'new Pi' site and flips to the 'Ps' site before the hydride transfer, was proposed. An alternative model based on a number of structures of B. stearothermophilus GAPDH ternary complexes (non-covalent and thioacyl intermediate) proposes that in the ternary Michaelis complex the C-3 phosphate binds to the 'Ps' site and flips from the 'Ps' to the 'new Pi' site during or after the redox step.     

"Half of the sites" binding of D-glyceraldehyde-3-phosphate dehydrogenase folding intermediate with GroEL

Two D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) folding intermediate subunits bind with chaperonin 60 (GroEL) to form a stable complex, which can no longer bind with additional GAPDH intermediate subunits, but does bind with one more lysozyme folding intermediate or one chaperonin 10 (GroES) molecule, suggesting that the two GAPDH subunits bind at one end of the GroEL molecule displaying a "half of the sites" binding profile. For lysozyme, GroEL binds with either one or two folding intermediates to form a stable 1:1 or 1:2 complex with one substrate on each end of the GroEL double ring for the latter. The 1:1 complex of GroEL.GroES binds with one lysozyme or one dimeric GAPDH folding intermediate to form a stable ternary complex. Both complexes of GroEL.lysozyme1 and GroEL.GAPDH2 bind with one GroES molecule only at the other end of the GroEL molecule forming a trans ternary complex. According to the stoichiometry of GroEL binding with the GAPDH folding intermediate and the formation of ternary complexes containing GroEL.GAPDH2, it is suggested that the folding intermediate of GAPDH binds, very likely in the dimeric form, with GroEL at one end only.     

The crystal structure of d-glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic archaeon Methanothermus fervidus in the presence of NADP(+) at 2.1 A resolution

The crystal structure of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the archaeon Methanothermus fervidus has been solved in the holo form at 2.1 A resolution by molecular replacement. Unlike bacterial and eukaryotic homologous enzymes which are strictly NAD(+)-dependent, GAPDH from this organism exhibits a dual-cofactor specificity, with a marked preference for NADP(+) over NAD(+). The present structure is the first archaeal GAPDH crystallized with NADP(+). GAPDH from M. fervidus adopts a homotetrameric quaternary structure which is topologically similar to that observed for its bacterial and eukaryotic counterparts. Within the cofactor-binding site, the positively charged side-chain of Lys33 decisively contributes to NADP(+) recognition through a tight electrostatic interaction with the adenosine 2'-phosphate group. Like other GAPDHs, GAPDH from archaeal sources binds the nicotinamide moiety of NADP(+) in a syn conformation with respect to the adjacent ribose and so belongs to the B-stereospecific class of oxidoreductases. Stabilization of the syn conformation is principally achieved through hydrogen bonding of the carboxamide group with the side-chain of Asp171, a structural feature clearly different from what is observed in all presently known GAPDHs from bacteria and eukaryotes. Within the catalytic site, the reported crystal structure definitively confirms the essential role previously assigned to Cys140 by site-directed mutagenesis studies. In conjunction with new mutation results reported in this paper, inspection of the crystal structure gives reliable evidence for the direct implication of the side-chain of His219 in the catalytic mechanism. M. fervidus grows optimally at 84 degrees C with a maximal growth temperature of 97 degrees C. The paper includes a detailed comparison of the present structure with four other homologous enzymes extracted from mesophilic as well as thermophilic organisms. Among the various phenomena related to protein thermostabilization, reinforcement of electrostatic and hydrophobic interactions as well as a more efficient molecular packing appear to be essentially promoted by the occurrence of two additional alpha-helices in the archaeal GAPDHs. The first one, named alpha4, is located in the catalytic domain and participates in the enzyme architecture at the quaternary structural level. The second one, named alphaJ, occurs at the C terminus and contributes to the molecular packing within each monomer by filling a peripherical pocket in the tetrameric assembly.     

Replacement of two invariant serine residues in chorismate synthase provides evidence that a proton relay system is essential for intermediate formation and catalytic activity

Chorismate synthase is the last enzyme of the common shikimate pathway, which catalyzes the anti-1,4-elimination of the 3-phosphate group and the C-(6proR) hydrogen from 5-enolpyruvylshikimate 3-phosphate (EPSP) to generate chorismate, a precursor for the biosynthesis of aromatic compounds. Enzyme activity relies on reduced FMN, which is thought to donate an electron transiently to the substrate, facilitating C(3)-O bond breakage. The crystal structure of the enzyme with bound EPSP and the flavin cofactor highlighted two invariant serine residues interacting with a bound water molecule that is close to the C(3)-O of EPSP. In this article we present the results of a mutagenesis study where we replaced the two invariant serine residues at positions 16 and 127 of the Neurospora crassa chorismate synthase with alanine, producing two single-mutant proteins (Ser16Ala and Ser127Ala) and a double-mutant protein (Ser16AlaSer127Ala). The residual activity of the Ser127Ala and Ser16Ala single-mutant proteins was found to be six-fold and 70-fold lower, respectively, than that of the wild-type protein. No residual activity was detected for the Ser16AlaSer127Ala double-mutant protein, and formation of the typical transient intermediate, characteristic for the chorismate synthase-catalysed reaction, was not observed, in contrast to the single-mutant proteins. On the basis of the structure of the enzyme, we propose that Ser16 and Ser127 form part of a proton relay system among the isoalloxazine ring of FMN, histidine 106 and the phosphate group of EPSP that is essential for the formation of the transient intermediate and for substrate turnover.     

Crystal structure of human muscle aldolase complexed with fructose 1,6-bisphosphate: mechanistic implications

Fructose 1,6-bisphosphate aldolase catalyzes the reversible cleavage of fructose 1,6-bisphosphate and fructose 1-phosphate to dihydroxyacetone phosphate and either glyceraldehyde 3-phosphate or glyceraldehyde, respectively. Catalysis involves the formation of a Schiff's base intermediate formed at the epsilon-amino group of Lys229. The existing apo-enzyme structure was refined using the crystallographic free-R-factor and maximum likelihood methods that have been shown to give improved structural results that are less subject to model bias. Crystals were also soaked with the natural substrate (fructose 1,6-bisphosphate), and the crystal structure of this complex has been determined to 2.8 A. The apo structure differs from the previous Brookhaven-deposited structure (1ald) in the flexible C-terminal region. This is also the region where the native and complex structures exhibit differences. The conformational changes between native and complex structure are not large, but the observed complex does not involve the full formation of the Schiff's base intermediate, and suggests a preliminary hydrogen-bonded Michaelis complex before the formation of the covalent complex.     

Roles of two conserved amino acid residues in the active site of galactose-1-phosphate uridylyltransferase: an essential serine and a nonessential cysteine

Galactose-1-phosphate uridylyltransferase (GalT) catalyzes the reversible transformation of uridine 5'-diphosphate glucose (UDPGlc) and galactose-1-phosphate into uridine 5'-diphosphate galactose (UDPGal) and glucose-1-phosphate through a double displacement mechanism, with the intermediate formation of a covalent uridylyl-enzyme (UMP-enzyme). The covalent linkage is a phosphoramidate formed between the UMP moiety and the His 166 N(epsilon)(2) of GalT, with His 166 N(delta1) retaining a proton throughout the catalytic cycle. Cys 160 and Ser 161 in Escherichia coli GalT are engaged in hydrogen bonding with the peripheral phosphoryl oxygen atoms of the substrate in the crystalline UMP-enzyme and in the crystalline complex of H166G-GalT with UDPGlc [Wedekind, J. E., Frey, P. A., and Rayment, I. (1996) Biochemistry 35, 11560-11569; Thoden, J. B., Ruzicka, F. J., Frey, P. A., Rayment, I., and Holden, H. M. (1997) Biochemistry 36, 1212-1222]. Site-directed mutagenesis, thermodynamic, transient kinetic, and steady-state kinetic studies have been performed to investigate the roles of Cys 160 and Ser 161 in catalysis. The absence of the thiol group of Cys 160 in the variants C160S and C160A did not seriously alter the enzymatic activity. However, the variant S161A displayed 7000-fold less activity than wild-type GalT. The low activity of S161A was directly related to impaired uridylylation rate constant (3.7 x 10(-)(2) s(-)(1)) and de-uridylylation rate constant (0.5 x 10(-)(2) s(-)(1)) resulting from a higher kinetic barrier for uridylyl-group transfer by the variant S161A as compared with the wild-type GalT. Equilibrium uridylylation studies showed that neither Cys 160 nor Ser 161 was involved in stabilizing the uridylyl-enzyme intermediate. The results lead to the conclusion that the conserved Cys 160 does not play a critical role in catalysis. Ser 161 is most likely involved in donating a hydrogen bond to the beta-phosphoryl group of a substrate, thereby providing proper orientation for nucleophilic catalysis.     

Glyceraldehyde-3-phosphate, a glycolytic intermediate, prevents cells from apoptosis by lowering S-nitrosylation of glyceraldehyde-3-phosphate dehydrogenase

Glyceraldehyde-3-phosphate (G-3-P), the substrate of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), is a key intermediate in several metabolic pathways. Recently, we reported that G-3-P directly inhibits caspase-3 activity in a reversible noncompetitive mode, suggesting the intracellular G-3-P level as a cell fate decision factor. It has been known that apoptotic stimuli induce the generation of NO, and NO S-nitrosylates GAPDH at the catalytic cysteine residue, which confers GAPDH the ability to bind to Siah-1, an E3 ubiquitin ligase. The GAPDH-Siah-1 complex is translocated into the nucleus and subsequently triggers the apoptotic process. Here, we clearly showed that intracellular G-3-P protects GAPDH from S-nitrosylation at above a certain level, and consequently maintains the cell survival. In case G-3-P drops below a certain level as a result of exposure to specific stimuli, G-3-P cannot inhibit S-nitrosylation of GAPDH anymore, and consequently GAPDH translocates with Siah-1 into the nucleus. Based on these results, we suggest that G-3-P functions as a molecule switch between cell survival and apoptosis by regulating S-nitrosylation of GAPDH.     

A stable cold folding intermediate of rabbit muscle D-glyceraldehyde 3-phosphate dehydrogenase.

With decreasing temperature the reactivation yield of denatured D-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) upon dilution increases but the reactivation rate decreases. Neither reactivation nor aggregation during refolding can be detected at 4 degrees C in 48 h, and at 3 degrees C even in 6 days. However, the reactivation takes place once the temperature is raised with little decrease of the yield after incubation for 6 days at 3 degrees C. A cold folding intermediate forms in a burst phase of refolding at 4 degrees C as shown by a fast change of the intrinsic fluorescence followed by further conformational adjustment to a stable state in about 1 h. The stable folding intermediate has been characterized to be a dimer of partially folded GAPDH subunit with secondary structure between that of the native and denatured enzymes, a hydrophobic cluster not found in either the native or the denatured state, and an active site similar to but different from that of the native state. Chaperonin 60 (GroEL) binds with all intermediates formed at 4 degrees C, but the intermediates formed at the early folding stage reactivate with higher yield than those formed after conformational adjustment when dissociated from GroEL in the presence of ATP and further folded and assembled into the native tetramer.     

Crystal structure of a phosphatase with a unique substrate binding domain from Thermotoga maritima

We have determined the crystal structure of a phosphatase with a unique substrate binding domain from Thermotoga maritima, TM0651 (gi 4981173), at 2.2 A resolution by selenomethionine single-wavelength anomalous diffraction (SAD) techniques. TM0651 is a member of the haloacid dehalogenase (HAD) superfamily, with sequence homology to trehalose-6-phosphate phosphatase and sucrose-6(F)-phosphate phosphohydrolase. Selenomethionine labeled TM0651 crystallized in space group C2 with three monomers per asymmetric unit. Each monomer has approximate dimensions of 65 x 40 x 35 A(3), and contains two domains: a domain of known hydrolase fold characteristic of the HAD family, and a domain with a new tertiary fold consisting of a six-stranded beta-sheet surrounded by four alpha-helices. There is one disulfide bond between residues Cys35 and Cys265 in each monomer. One magnesium ion and one sulfate ion are bound in the active site. The superposition of active site residues with other HAD family members indicates that TM0651 is very likely a phosphatase that acts through the formation of a phosphoaspartate intermediate, which is supported by both NMR titration data and a biochemical assay. Structural and functional database searches and the presence of many aromatic residues in the interface of the two domains suggest the substrate of TM0651 is a carbohydrate molecule. From the crystal structure and NMR data, the protein likely undergoes a conformational change upon substrate binding.     

Oxyanion hole-stabilized stereospecific isomerization in ribose-5-phosphate isomerase (Rpi)

Ribose-5-phosphate isomerase (Rpi) acts as a key enzyme in the oxidative and reductive pentose-phosphate pathways for the conversion of ribose-5-phosphate (R5P) to ribulose-5-phosphate and vice versa. We have determined the crystal structures of Rpi from Thermus thermophilus HB8 in complex with the open chain form of the substrate R5P and the open chain form of the C2 epimeric inhibitor arabinose-5-phosphate as well as the apo form at high resolution. The crystal structures of both complexes revealed that these ring-opened epimers are bound in the active site in a mirror symmetry binding mode. The O1 atoms are stabilized by an oxyanion hole composed of the backbone amide nitrogens in the conserved motif. In the structure of the Rpi.R5P complex, the conversion moiety O1-C1-C2-O2 in cis-configuration interacts with the carboxyl oxygens of Glu-108 in a water-excluded environment. Furthermore, the C2 hydroxyl group is presumed to be highly polarized by short hydrogen bonding with the side chain of Lys-99. R5P bound as the ring-opened reaction intermediate clarified the high stereoselectivity of the catalysis and is consistent with an aldose-ketose conversion by Rpi that proceeds via a cis-enediolate intermediate.