Cooperative binding of gamma-glutamyl substrate to human glutathione synthetase.

Human glutathione synthetase is responsible for catalyzing the final step in glutathione biosynthesis. It is a homodimer with a monomer subunit MW of 52 kDa. Kinetic analysis reveals a departure from linearity of the Lineweaver-Burk double reciprocal plot for the binding of gamma-glutamyl substrate, indicating cooperative binding. The measured apparent K(m) values for gamma-glutamyl-alpha-aminobutyrate (an analog of gamma-glutamyl-alpha-aminobutyrate) are 63 and 164 microM, respectively. Neither ATP (K(m) of 248 microM) nor glycine (K(m) of 452 microM) exhibits such cooperative binding behavior. Although ATP is proposed to play a key role in the sequential binding of gamma-glutamyl substrate to the enzyme, the cooperative binding of the gamma-glutamyl substrate is not affected by alterations of ATP concentration. Quantitative analysis of the kinetic results for gamma-glutamyl substrate binding gives a Hill coefficient (h) of 0.75, indicating negative cooperativity. Our studies, for the first time, show that human glutathione synthetase is an allosteric enzyme with cooperative binding for gamma-glutamyl substrate.     

Mutational substitution of residues implicated by crystal structure in binding the substrate glutathione to human glutathione S-transferase pi.

Site-directed substitution mutations were introduced into a cDNA expression vector (pUC120 pi) that encoded a human glutathione S-transferase pi isozyme to non-conservatively replace four residues (Tyr7, Arg13, Gln62 and Asp96). Our earlier X-ray crystallographic analysis implicated these residues in binding and/or chemically activating the substrate glutathione. Each substitution mutation decreased the specific activity of the enzyme to less than 2% of the wild-type. Glutathione-binding was also reduced; however, the Tyr7----Phe mutant still retained 27% of the wild-type capacity to bind glutathione, underlining the primary role that this residue is likely to play in chemically activating the glutathione molecule during catalysis.     

Novel kinetics of mammalian glutathione synthetase: characterization of gamma-glutamyl substrate cooperative binding

Glutathione (GSH) synthetase [L-gamma-glutamyl-L-cysteinyl:glycine ligase (ADP-forming), EC 6.3.2.3] catalyzes the final step in GSH biosynthesis. Mammalian glutathione synthetase is a homodimer with each subunit containing an active site. We report the detailed kinetic data for purified recombinant rat glutathione synthetase. It has the highest specific activity (11 micromol/min/mg) reported for any mammalian glutathione synthetase. The apparent K(m) values for ATP and glycine are 37 and 913 microM, respectively. The Lineweaver-Burk double reciprocal plot for gamma-glutamyl substrate binding revealed a departure from linearity indicating cooperative binding. Quantitative analysis of the kinetic results for gamma-glutamyl substrate binding gives a Hill coefficient (h) of 0. 576, which shows the negative cooperativity. Neither ATP, the other substrate involved in forming the enzyme-bound gamma-glutamyl phosphate intermediate, nor glycine, which attacks this intermediate to form GSH, exhibit any cooperativity. The cooperative binding of gamma-glutamyl substrate is not affected by ATP concentration. Thus, mammalian glutathione synthetase is an allosteric enzyme.     

Ebola virus glycoprotein 1: identification of residues important for binding and postbinding events

The filoviruses Ebola virus (EBOV) and Marburg virus (MARV) are responsible for devastating hemorrhagic fever outbreaks. No therapies are available against these viruses. An understanding of filoviral glycoprotein 1 (GP1) residues involved in entry events would facilitate the development of antivirals. Towards this end, we performed alanine scanning mutagenesis on selected residues in the amino terminus of GP1. Mutant GPs were evaluated for their incorporation onto feline immunodeficiency virus (FIV) particles, transduction efficiency, receptor binding, and ability to be cleaved by cathepsins L and B. FIV virions bearing 39 out of 63 mutant glycoproteins transduced cells efficiently, whereas virions bearing the other 24 had reduced levels of transduction. Virions pseudotyped with 23 of the poorly transducing GPs were characterized for their block in entry. Ten mutant GPs were very poorly incorporated onto viral particles. Nine additional mutant GPs (G87A/F88A, K114A/K115A, K140A, G143A, P146A/C147A, F153A/H154A, F159A, F160A, and Y162A) competed poorly with wild-type GP for binding to permissive cells. Four of these nine mutants (P146A/C147A, F153A/H154A, F159A, and F160A) were also inefficiently cleaved by cathepsins. An additional four mutant GPs (K84A, R134A, D150A, and E305/E306A) that were partially defective in transduction were found to compete effectively for receptor binding and were readily cleaved by cathepsins. This finding suggested that this latter group of mutants might be defective at a postbinding, cathepsin cleavage-independent step. In total, our study confirms the role of some GP1 residues in EBOV entry that had previously been recognized and identifies for the first time other residues that are important for productive entry.     

Aspartate 458 of human glutathione synthetase is important for cooperativity and active site structure

Human glutathione synthetase (hGS) catalyzes the second ATP-dependent step in the biosynthesis of glutathione (GSH) and is negatively cooperative to the γ-glutamyl substrate. The hGS active site is composed of three highly conserved catalytic loops, notably the alanine rich A-loop. Experimental and computational investigations of the impact of mutation of Asp458 are reported, and thus the role of this A-loop residue on hGS structure, activity, negativity cooperativity and stability is defined. Several Asp458 hGS mutants (D458A, D458N and D458R) were constructed using site-directed mutagenesis and their activities determined (10%, 15% and 7% of wild-type hGS, respectively). The Michaelis–Menten constant (K_m) was determined for all three substrates (glycine, GAB and ATP): glycine K_m increased by 30–115-fold, GAB K_m decreased by 8–17-fold, and the ATP K_m was unchanged. All Asp458 mutants display a change in cooperativity from negative cooperativity to non-cooperative. All mutants show similar stability as compared to wild-type hGS, as determined by differential scanning calorimetry. The findings indicate that Asp458 is essential for hGS catalysis and that it impacts the allostery of hGS.     

Leishmania major thialysine Nepsilon-acetyltransferase: identification of amino acid residues crucial for substrate binding

Thialysine N(epsilon)-acetyltransferases and spermidine/spermine N-acetyltransferases (SSAT) are closely related members of the GCN5-related N-acetyltransferase superfamily. Accordingly, a putative orthologue from the human protozoan parasite Leishmania major exhibits an almost equal similarity to human SSAT and thialysine N(epsilon)-acetyltransferase. Characterisation of the recombinantly expressed L. major protein indicated that it represents a thialysine N(epsilon)-acetyltransferase, preferring thialysine (S-aminoethyl-l-cysteine) and structurally related amino acids as acceptor molecules. The known thialysine N(epsilon)-acetyltransferases contain five conserved amino acid residues that are replaced in SSAT sequences. Kinetic analyses of the respective recombinant mutant proteins suggest that Ser(82) and Thr(83) of L. major thialysine N(epsilon)-acetyltransferase are key residues for acceptor binding. In addition, the conserved Leu(130) is tentatively involved in specific interaction with the sulphur-containing side chain of thialysine. The presence of these three amino acid residues is suggested to be a means by which thialysine N(epsilon)-acetyltransferases can be distinguished from SSAT sequences.     

The crystal structure of human PAPS synthetase 1 reveals asymmetry in substrate binding

The high energy sulfate donor 3'-phosphoadenosine-5-phosphosulfate (PAPS) is used for sulfate conjugation of extracellular matrix, hormones and drugs. Human PAPS synthetase 1 catalyzes two subsequent reactions starting from ATP and sulfate. First the ATP sulfurylase domain forms APS, then the APS kinase domain phosphorylates the APS intermediate to PAPS. Up to now the interaction between the two enzymatic activities remained elusive, mainly because of missing structural information. Here we present the crystal structure of human PAPSS1 at 1.8 angstroms resolution. The structure reveals a homodimeric, asymmetric complex with the shape of a chair. The two kinase domains adopt different conformational states, with only one being able to bind its two substrates. The asymmetric binding of ADP to the APS kinase is not only observed in the crystal structure, but can also be detected in solution, using an enzymatic assay. These observations strongly indicate structural changes during the reaction cycle. Furthermore crystals soaked with ADP and APS could be prepared and the corresponding structures could be solved.     

Selectivity and specificity of substrate binding in methionyl-tRNA synthetase

The accuracy of in vivo incorporation of amino acids during protein biosynthesis is controlled to a significant extent by aminoacyl-tRNA synthetases (aaRS). This paper describes the application of the HierDock computational method to study the molecular basis of amino acid binding to the Escherichia coli methionyl tRNA synthetase (MetRS). Starting with the protein structure from the MetRS cocrystal, the HierDock calculations predict the binding site of methionine in MetRS to a root mean square deviation in coordinates (CRMS) of 0.55 A for all the atoms, compared with the crystal structure. The MetRS conformation in the cocrystal structure shows good discrimination between cognate and the 19 noncognate amino acids. In addition, the calculated binding energies of a set of five methionine analogs show a good correlation (R(2) = 0.86) to the relative free energies of binding derived from the measured in vitro kinetic parameters, K(m) and k(cat). Starting with the crystal structure of MetRS without the methionine (apo-MetRS), the putative binding site of methionine was predicted. We demonstrate that even the apo-MetRS structure shows a preference for binding methionine compared with the 19 other natural amino acids. On comparing the calculated binding energies of the 20 natural amino acids for apo-MetRS with those for the cocrystal structure, we observe that the discrimination against the noncognate substrate increases dramatically in the second step of the physical binding process associated with the conformation change in the protein.     

Homology of Escherichia coli B glutathione synthetase with dihydrofolate reductase in amino acid sequence and substrate binding site

Glutathione synthetase from Escherichia coli B showed amino acid sequence homology with mammalian and bacterial dihydrofolate reductases over 40 residues, although these two enzymes are different in their reaction mechanisms and ligand requirements. The effects of ligands of dihydrofolate reductase on the reaction of E. coli B glutathione synthetase were examined to find resemblances in catalytic function to dihydrofolate reductase. The E. coli B enzyme was potently inhibited by 7,8-dihydrofolate, methotrexate, and trimethoprim. Methotrexate was studied in detail and proved to bind to an ATP binding site of the E. coli B enzyme with K1 value of 0.1 mM. The homologous portion of the amino acid sequence in dihydrofolate reductases, which corresponds to the portion coded by exon 3 of mammalian dihydrofolate reductase genes, provided a binding site of the adenosine diphosphate moiety of NADPH in the crystal structure of dihydrofolate reductase. These analyses would indicate that the homologous portion of the amino acid sequence of the E. coli B enzyme provides the ATP binding site. This report gives experimental evidence that amino acid sequences related by sequence homology conserve functional similarity even in enzymes which differ in their catalytic mechanisms.     

Characterization of putative hydrophobic substrate binding site residues of a Delta class glutathione transferase from Anopheles dirus

To date, investigations of the hydrophobic substrate site of the insect Delta class glutathione transferase are limited in number. In the present study, putative hydrophobic site residues of AdGSTD4-4 have been proposed and characterized. These residues are Gln-112, Thr-174, Phe-212, Arg-214, Tyr-215 and Phe-216. It was found that Gln-112 does not contribute significantly to the catalytic properties of AdGSTD4-4. Arg-214, Tyr-215 and Phe-216 made contributions to catalytic properties and the rate-limiting step. Thr-174 and Phe-212 appeared to be important in enzymatic catalysis by stabilizing the active site β1-α1 loop on which the critical catalytic residue Ser-9 is located. The aromatic Phe-212 pi cloud appears to be important for interactions with its hydrophobic size representing an almost equally important factor. The data suggests that these residues are not directly involved in catalysis but exert their influence through secondary interactions. In addition, active site rearrangements occur to bring different residues into play even for conjugation through the same mechanisms. Therefore, due to the conformational rearrangements topologically equivalent residues observed in crystal structures may not perform equivalent roles in catalysis in different GST classes.     

Function of conserved residues of human glutathione synthetase: implications for the ATP-grasp enzymes

Glutathione synthetase is an enzyme that belongs to the glutathione synthetase ATP-binding domain-like superfamily. It catalyzes the second step in the biosynthesis of glutathione from gamma-glutamylcysteine and glycine in an ATP-dependent manner. Glutathione synthetase has been purified and sequenced from a variety of biological sources; still, its exact mechanism is not fully understood. A variety of structural alignment methods were applied and four highly conserved residues of human glutathione synthetase (Glu-144, Asn-146, Lys-305, and Lys-364) were identified in the binding site. The function of these was studied by experimental and computational site-directed mutagenesis. The three-dimensional coordinates for several human glutathione synthetase mutant enzymes were obtained using molecular mechanics and molecular dynamics simulation techniques, starting from the reported crystal structure of human glutathione synthetase. Consistent with circular dichroism spectroscopy, our results showed no major changes to overall enzyme structure upon residue mutation. However, semiempirical calculations revealed that ligand binding is affected by these mutations. The key interactions between conserved residues and ligands were detected and found to be essential for enzymatic activity. Particularly, the negatively charged Glu-144 residue plays a major role in catalysis.     

2.9 A crystal structure of ligand-free tryptophanyl-tRNA synthetase: domain movements fragment the adenine nucleotide binding site

The crystal structure of ligand-free tryptophanyl-tRNA synthetase (TrpRS) was solved at 2.9 A using a combination of molecular replacement and maximum-entropy map/phase improvement. The dimeric structure (R = 23.7, Rfree = 26.2) is asymmetric, unlike that of the TrpRS tryptophanyl-5'AMP complex (TAM; Doublié S, Bricogne G, Gilmore CJ, Carter CW Jr, 1995, Structure 3:17-31). In agreement with small-angle solution X-ray scattering experiments, unliganded TrpRS has a conformation in which both monomers open, leaving only the tryptophan-binding regions of their active sites intact. The amino terminal alphaA-helix, TIGN, and KMSKS signature sequences, and the distal helical domain rotate as a single rigid body away from the dinucleotide-binding fold domain, opening the AMP binding site, seen in the TAM complex, into two halves. Comparison of side-chain packing in ligand-free TrpRS and the TAM complex, using identification of nonpolar nuclei (Ilyin VA, 1994, Protein Eng 7:1189-1195), shows that significant repacking occurs between three relatively stable core regions, one of which acts as a bearing between the other two. These domain rearrangements provide a new structural paradigm that is consistent in detail with the "induced-fit" mechanism proposed for TyrRS by Fersht et al. (Fersht AR, Knill-Jones JW, Beduelle H, Winter G, 1988, Biochemistry 27:1581-1587). Coupling of ATP binding determinants associated with the two catalytic signature sequences to the helical domain containing the presumptive anticodon-binding site provides a mechanism to coordinate active-site chemistry with relocation of the major tRNA binding determinants.     

Crystal structure of mannanase 26A from Pseudomonas cellulosa and analysis of residues involved in substrate binding

The crystal structure of Pseudomonas cellulosa mannanase 26A has been solved by multiple isomorphous replacement and refined at 1.85 A resolution to an R-factor of 0.182 (R-free = 0.211). The enzyme comprises (beta/alpha)(8)-barrel architecture with two catalytic glutamates at the ends of beta-strands 4 and 7 in precisely the same location as the corresponding glutamates in other 4/7-superfamily glycoside hydrolase enzymes (clan GH-A glycoside hydrolases). The family 26 glycoside hydrolases are therefore members of clan GH-A. Functional analyses of mannanase 26A, informed by the crystal structure of the enzyme, provided important insights into the role of residues close to the catalytic glutamates. These data showed that Trp-360 played a critical role in binding substrate at the -1 subsite, whereas Tyr-285 was important to the function of the nucleophile catalyst. His-211 in mannanase 26A does not have the same function as the equivalent asparagine in the other GH-A enzymes. The data also suggest that Trp-217 and Trp-162 are important for the activity of mannanase 26A against mannooligosaccharides but are less important for activity against polysaccharides.     

The structure of Escherichia coli ATP-phosphoribosyltransferase: identification of substrate binding sites and mode of AMP inhibition

ATP-phosphoribosyltransferase (ATP-PRT), the first enzyme of the histidine pathway, is a complex allosterically regulated enzyme, which controls the flow of intermediates through this biosynthetic pathway. The crystal structures of Escherichia coli ATP-PRT have been solved in complex with the inhibitor AMP at 2.7A and with product PR-ATP at 2.9A (the ribosyl-triphosphate could not be resolved). On the basis of binding of AMP and PR-ATP and comparison with type I PRTs, the PRPP and parts of the ATP-binding site are identified. These structures clearly identify the AMP as binding in the 5-phosphoribosyl-alpha-1-pyrophosphate (PRPP)-binding site, with the adenosine ring occupying the ATP-binding site. Comparison with the recently solved Mycobacterium tuberculosis ATP-PRT structures indicates that histidine is solely responsible for the large conformational changes observed between the hexameric forms of the enzyme. The role of oligomerisation in inhibition and the structural basis for the synergistic inhibition by histidine and AMP are discussed.     

Structural studies of lysyl-tRNA synthetase: conformational changes induced by substrate binding

Lysyl-tRNA synthetase is a member of the class II aminoacyl-tRNA synthetases and catalyses the specific aminoacylation of tRNA(Lys). The crystal structure of the constitutive lysyl-tRNA synthetase (LysS) from Escherichia coli has been determined to 2.7 A resolution in the unliganded form and in a complex with the lysine substrate. A comparison between the unliganded and lysine-bound structures reveals major conformational changes upon lysine binding. The lysine substrate is involved in a network of hydrogen bonds. Two of these interactions, one between the alpha-amino group and the carbonyl oxygen of Gly 216 and the other between the carboxylate group and the side chain of Arg 262, trigger a subtle and complicated reorganization of the active site, involving the ordering of two loops (residues 215-217 and 444-455), a change in conformation of residues 393-409, and a rotation of a 4-helix bundle domain (located between motif 2 and 3) by 10 degrees. The result of these changes is a closing up of the active site upon lysine binding.     

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.     

Reaction mechanism of glutathione synthetase from Arabidopsis thaliana: site-directed mutagenesis of active site residues

Glutathione is essential for maintaining the intracellular redox environment and is synthesized from gamma-glutamylcysteine, glycine, and ATP by glutathione synthetase (GS). To examine the reaction mechanism of a eukaryotic GS, 24 Arabidopsis thaliana GS (AtGS) mutants were kinetically characterized. Within the gamma-glutamylcysteine/glutathione-binding site, the S153A and S155A mutants displayed less than 4-fold changes in kinetic parameters with mutations of Glu-220 (E220A/E220Q), Gln-226 (Q226A/Q226N), and Arg-274 (R274A/R274K) at the distal end of the binding site resulting in 24-180-fold increases in the K(m) values for gamma-glutamylcysteine. Substitution of multiple residues interacting with ATP (K313M, K367M, and E429A/E429Q) or coordinating magnesium ions to ATP (E148A/E148Q, N150A/N150D, and E371A) yielded inactive protein because of compromised nucleotide binding, as determined by fluorescence titration. Other mutations in the ATP-binding site (E371Q, N376A, and K456M) resulted in greater than 30-fold decreases in affinity for ATP and up to 80-fold reductions in turnover rate. Mutation of Arg-132 and Arg-454, which are positioned at the interface of the two substrate-binding sites, affected the enzymatic activity differently. The R132A mutant was inactive, and the R132K mutant decreased k(cat) by 200-fold; however, both mutants bound ATP with K(d) values similar to wild-type enzyme. Minimal changes in kinetic parameters were observed with the R454K mutant, but the R454A mutant displayed a 160-fold decrease in k(cat). In addition, the R132K, R454A, and R454K mutations elevated the K(m) value for glycine up to 11-fold. Comparison of the pH profiles and the solvent deuterium isotope effects of A. thaliana GS and the Arg-132 and Arg-454 mutants also suggest distinct mechanistic roles for these residues. Based on these results, a catalytic mechanism for the eukaryotic GS is proposed.