The bifunctional active site of S-adenosylmethionine synthetase. Roles of the basic residues

S-adenosylmethionine (AdoMet) synthetase catalyzes a unique two-step enzymatic reaction leading to formation of the primary biological alkylating agent. The crystal structure of Escherichia coli AdoMet synthetase shows that the active site, which lies between two subunits, contains four lysines and one histidine as basic residues. In order to test the proposed charge and hydrogen bonding roles in catalytic function, each lysine has been changed to an uncharged methionine or alanine, and the histidine has been altered to asparagine. The resultant enzyme variants are all tetramers like the wild type enzyme; however, circular dichroism spectra show reductions in helix content for the K245*M and K269M mutants. (The asterisk denotes that the residue is in the second subunit.) Four mutants have k(cat) reductions of approximately 10(3)-10(4)-fold in AdoMet synthesis; however, the k(cat) of K165*M variant is only reduced 2-fold. In each mutant, there is a smaller catalytic impairment in the partial reaction of tripolyphosphate hydrolysis. The K165*A enzyme has a 100-fold greater k(cat) for tripolyphosphate hydrolysis than the wild type enzyme, but this mutant is not activated by AdoMet in contrast to the wild type enzyme. The properties of these mutants require reassessment of the catalytic roles of these residues.     

The active site loop of S-adenosylmethionine synthetase modulates catalytic efficiency

Crystallographic studies of Escherichia coli S-adenosylmethionine synthetase (ATP:L-methionine S-adenosyltransferase, MAT) have defined a flexible polypeptide loop that can gate access to the active site without contacting the substrates. The influence of the length and sequence of this active site loop on catalytic efficiency has been characterized in a mutant in which the E. coli MAT sequence (DRADPLEQ) has been replaced with the distinct sequence of the corresponding region of the otherwise highly homologous rat liver enzyme (HDLRNEEDV). Four additional mutants in which the entire DRADPLEQ sequence was replaced by five, six, seven, or eight glycines have been studied to unveil the effects of loop length and the influence of side chains. In all of the mutants, the maximal rate of S-adenosylmethionine formation (k(cat)) is diminished by more than 200-fold whereas the rate of hydrolysis of the tripolyphosphate intermediate is decreased by less than 3-fold. Thus, the function of the loop is localized to the first step in the overall reaction. The K(m) for methionine increases in all of the oligoglycine mutants, whereas the K(m) values for ATP are not substantially different. The k(cat) for the wild-type enzyme is decreased by increases in solution microviscosity with 55% of the maximal dependence. Thus, a diffusional event is coupled to the chemical step of AdoMet formation, which is known to be rate-limiting. The results indicate that a conformational change, possibly loop closure, is associated with AdoMet synthesis. The data integrate a previously discovered conformational change associated with PPP(i) binding to the E x AdoMet complex into the reaction sequence, reflecting a difference in protein conformation in the E x AdoMet x PPP(i) complex whether it is formed from the E x ATP x methionine complex or from binding of exogenous PPP(i). The temperature dependence of the k(cat) for S-adenosylmethionine formation shows that the removal of the side chains in the glycine mutants causes the activation enthalpy of the reaction to approximately double in each case, while the activation entropy changes from negative in the wild-type enzyme to positive in the mutants. The favorable activation entropy in the mutant-catalyzed reactions may reflect release of water during catalysis, while the negative activation entropy in the reaction catalyzed by the wild-type enzyme apparently reflects reorganization of the loop. The observations point to how nature can fine-tune the activity of an enzyme by modifying substrate and product access to the active site rather than by altering the enzyme x substrate contacts or the catalytic machinery itself.     

Identification of active site residues in Bradyrhizobium japonicum acetyl-CoA synthetase.

Acetyl-CoA synthetase (ACS) catalyses the activation of acetate to acetyl-CoA in the presence of ATP and CoA. The gene encoding Bradyrhyzobium japonicum ACS has been cloned, sequenced, and expressed in Escherichia coli. The enzyme comprises 648 amino acid residues with a calculated molecular mass of 71,996 Da. The recombinant enzyme was also purified from the transformed E. coli. The enzyme was essentially indistinguishable from the ACS of B. japonicum bacteroids as to the criteria of polyacrylamide gel electrophoresis and biochemical properties. Based on the results of database analysis, Gly-263, Gly-266, Lys-269, and Glu-414 were selected for site-directed mutagenesis in order to identify amino acid residues essential for substrate binding and/or catalysis. Four different mutant enzymes (G263I, G266I, K269G, and E414Q) were prepared and then subjected to steady-state kinetic studies. The kinetic data obtained for the mutants suggest that Gly-266 and Lys-269 participate in the formation of acetyl-AMP, whereas Glu-414 may play a role in acetate binding.     

Ammonium scanning in an enzyme active site. The chiral specificity of aspartyl-tRNA synthetase

D-amino acids are largely excluded from protein synthesis, yet they are of great interest in biotechnology. Aspartyl-tRNA synthetase (AspRS) can misacylate tRNA(Asp) with D-aspartate instead of its usual substrate, L-Asp. We investigate how the preference for L-Asp arises, using molecular dynamics simulations. Asp presents a special problem, having pseudosymmetry broken only by its ammonium group, and AspRS must protect not only against D-Asp, but against an "inverted" orientation where the two substrate carboxylates are swapped. We compare L-Asp and D-Asp, in both orientations, and succinate, where the ammonium group is removed and the ligand has an additional negative charge. All possible ammonium positions on the ligand are thus scanned, providing information on electrostatic interactions. As controls, we simulate a Q199E mutation, obtaining a reduction in binding free energy in agreement with experiment, and we simulate TyrRS, which can misacylate tRNA(Tyr) with D-Tyr. For both TyrRS and AspRS, we obtain a moderate binding free energy difference DeltaDeltaG between the L- and D-amino acids, in agreement with their known ability to misacylate their tRNAs. In contrast, we predict that AspRS is strongly protected against inverted L-Asp binding. For succinate, kinetic measurements reveal a DeltaDeltaG of over 5 kcal/mol, favoring L-Asp. The simulations show how chiral discriminations arises from the structures, with two AspRS conformations acting in different ways and proton uptake by nearby histidines playing a role. A complex network of charges protects AspRS against most binding errors, making the engineering of its specificity a difficult challenge.     

Structure of the E. coli bifunctional GlmU acetyltransferase active site with substrates and products

The biosynthesis of UDP-GlcNAc in bacteria is carried out by GlmU, an essential bifunctional uridyltransferase that catalyzes the CoA-dependent acetylation of GlcN-1-PO(4) to form GlcNAc-1-PO(4) and its subsequent condensation with UTP to form pyrophosphate and UDP-GlcNAc. As a metabolite, UDP-GlcNAc is situated at a branch point leading to the biosynthesis of lipopolysaccharide and peptidoglycan. Consequently, GlmU is regarded as an important target for potential antibacterial agents. The crystal structure of the Escherichia coli GlmU acetyltransferase active site has been determined in complexes with acetyl-CoA, CoA/GlcN-1-PO(4), and desulpho-CoA/GlcNAc-1-PO(4). These structures reveal the enzyme groups responsible for binding the substrates. A superposition of these complex structures suggests that the 2-amino group of GlcN-1-PO(4) is positioned in proximity to the acetyl-CoA to facilitate direct attack on its thioester by a ternary complex mechanism.     

The active site of yeast aspartyl-tRNA synthetase: structural and functional aspects of the aminoacylation reaction.

The crystal structures of the various complexes formed by yeast aspartyl-tRNA synthetase (AspRS) and its substrates provide snapshots of the active site corresponding to different steps of the aminoacylation reaction. Native crystals of the binary complex tRNA-AspRS were soaked in solutions containing the two other substrates, ATP (or its analog AMPPcP) and aspartic acid. When all substrates are present in the crystal, this leads to the formation of the aspartyl-adenylate and/or the aspartyl-tRNA. A class II-specific pathway for the aminoacylation reaction is proposed which explains the known functional differences between the two classes while preserving a common framework. Extended signature sequences characteristic of class II aaRS (motifs 2 and 3) constitute the basic functional unit. The ATP molecule adopts a bent conformation, stabilized by the invariant Arg531 of motif 3 and a magnesium ion coordinated to the pyrophosphate group and to two class-invariant acidic residues. The aspartic acid substrate is positioned by a class II invariant acidic residue, Asp342, interacting with the amino group and by amino acids conserved in the aspartyl synthetase family. The amino acids in contact with the substrates have been probed by site-directed mutagenesis for their functional implication.     

Effectors of the stringent response target the active site of Escherichia coli adenylosuccinate synthetase.

Guanosine 5'-diphosphate 3'-diphosphate (ppGpp), a pleiotropic effector of the stringent response, potently inhibits adenylosuccinate synthetase from Escherichia coli as an allosteric effector and/or as a competitive inhibitor with respect to GTP. Crystals of the synthetase grown in the presence of IMP, hadacidin, NO3-, and Mg2+, then soaked with ppGpp, reveal electron density at the GTP pocket which is consistent with guanosine 5'-diphosphate 2':3'-cyclic monophosphate. Unlike ligand complexes of the synthetase involving IMP and GDP, the coordination of Mg2+ in this complex is octahedral with the side chain of Asp13 in the inner sphere of the cation. The cyclic phosphoryl group interacts directly with the side chain of Lys49 and indirectly through bridging water molecules with the side chains of Asn295 and Arg305. The synthetase either directly facilitates the formation of the cyclic nucleotide or scavenges trace amounts of the cyclic nucleotide from solution. Regardless of its mode of generation, the cyclic nucleotide binds far more tightly to the active site than does ppGpp. Conceivably, synthetase activity in vivo during the stringent response may be sensitive to the relative concentrations of several effectors, which together exercise precise control over the de novo synthesis of AMP.     

The mitochondrial tyrosyl-tRNA synthetase of Podospora anserina is a bifunctional enzyme active in protein synthesis and RNA splicing.

The Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (mt tyrRS), which is encoded by the nuclear gene cyt-18, functions not only in aminoacylation but also in the splicing of group I introns. Here, we isolated the cognate Podospora anserina mt tyrRS gene, designated yts1, by using the N. crassa cyt-18 gene as a hybridization probe. DNA sequencing of the P. anserina gene revealed an open reading frame (ORF) of 641 amino acids which has significant similarity to other tyrRSs. The yts1 ORF is interrupted by two introns, one near its N terminus at the same position as the single intron in the cyt-18 gene and the other downstream in a region corresponding to the nucleotide-binding fold. The P. anserina yts1+ gene transformed the N. crassa cyt-18-2 mutant at a high frequency and rescued both the splicing and protein synthesis defects. Furthermore, the YTS1 protein synthesized in Escherichia coli was capable of splicing the N. crassa mt large rRNA intron in vitro. Together, these results indicate that YTS1 is a bifunctional protein active in both splicing and protein synthesis. The P. anserina YTS1 and N. crassa CYT-18 proteins share three blocks of amino acids that are not conserved in bacterial or yeast mt tyrRSs which do not function in splicing. One of these blocks corresponds to the idiosyncratic N-terminal domain shown previously to be required for splicing activity of the CYT-18 protein. The other two are located in the putative tRNA-binding domain toward the C terminus of the protein and also appear to be required for splicing. Since the E. coli and yeast mt tyrRSs do not function in splicing, the adaptation of the Neurospora and Podospora spp. mt tyrRSs to function in splicing most likely occurred after the divergence of their common ancestor from yeast.     

Clostridium pasteurianum glutamine synthetase mechanism. Evidence for active site tyrosine residues

Preliminary chemical modification studies indicated the presence of tyrosine, carboxyl, arginine, histidine and the absence of serine and sulfhydryl residues at or near the active site of Clostridium pasteurianum glutamine synthetase. The conditions for tyrosine modification with tetranitromethane were optimized. The inactivation kinetics follow pseudo-first-order kinetics with respect to enzyme and second order with respect to modifier per active site. There was no inactivation at pH 6.5 suggesting the absence of thiol oxidation. The synthetase and transferase reactions followed the same pattern of inactivation on enzyme modification and both were equally protected by glutamate plus ATP. Thus tyrosine residues are present at the active site of the enzyme and are essential for both transferase and synthetase activities.     

Labeling of specific lysine residues at the active site of glutamine synthetase

Glutamine synthetase (Escherichia coli) was incubated with three different reagents that react with lysine residues, viz. pyridoxal phosphate, 5'-p-fluorosulfonylbenzoyladenosine, and thiourea dioxide. The latter reagent reacts with the epsilon-nitrogen of lysine to produce homoarginine as shown by amino acid analysis, nmr, and mass spectral analysis of the products. A variety of differential labeling experiments were conducted with the above three reagents to label specific lysine residues. Thus pyridoxal phosphate was found to modify 2 lysine residues leading to an alteration of catalytic activity. At least 1 lysine residue has been reported previously to be modified by pyridoxal phosphate at the active site of glutamine synthetase (Whitley, E. J., and Ginsburg, A. (1978) J. Biol. Chem. 253, 7017-7025). By varying the pH and buffer, one or both residues could be modified. One of these lysine residues was associated with approximately 81% loss in activity after modification while modification of the second lysine residue led to complete inactivation of the enzyme. This second lysine was found to be the residue which reacted specifically with the ATP affinity label 5'-p-fluorosulfonylbenzoyladenosine. Lys-47 has been previously identified as the residue that reacts with this reagent (Pinkofsky, H. B., Ginsburg, A., Reardon, I., Heinrikson, R. L. (1984) J. Biol. Chem. 259, 9616-9622; Foster, W. B., Griffith, M. J., and Kingdon, H. S. (1981) J. Biol. Chem. 256, 882-886). Thiourea dioxide inactivated glutamine synthetase with total loss of activity and concomitant modification of a single lysine residue. The modified amino acid was identified as homoarginine by amino acid analysis. The lysine residue modified by thiourea dioxide was established by differential labeling experiments to be the same residue associated with the 81% partial loss of activity upon pyridoxal phosphate inactivation. Inactivation with either thiourea dioxide or pyridoxal phosphate did not affect ATP binding but glutamate binding was weakened. The glutamate site was implicated as the site of thiourea dioxide modification based on protection against inactivation by saturating levels of glutamate. Glutamate also protected against pyridoxal phosphate labeling of the lysine consistent with this residue being the common site of reaction with thiourea dioxide and pyridoxal phosphate.     

Ancient adaptation of the active site of tryptophanyl-tRNA synthetase for tryptophan binding

The amino acid binding domains of the tryptophanyl (TrpRS)- and tyrosyl-tRNA synthetases (TyrRS) of Bacillus stearothermophilus are highly homologous. These similarities suggest that conserved residues in TrpRS may be responsible for both determining tryptophan recognition and discrimination against tyrosine. This was investigated by the systematic mutation of TrpRS residues based upon the identity of homologous positions in TyrRS. Of the four residues which interact directly with the aromatic side chain of tryptophan (Phe5, Met129, Asp132, and Val141) replacements of Asp132 led to significant changes in the catalytic efficiency of Trp aminoacylation (200-1250-fold reduction in k(cat)/K(M)) and substitution of Val141 by the larger Glu side chain reduced k(cat)/K(M) by 300-fold. Mutation of Pro127, which determines the position of active-site residues, did not significantly effect Trp binding. Of the mutants tested, D132N TrpRS also showed a significant reduction in discrimination against Tyr, with Tyr acting as a competitive inhibitor but not a substrate. The analogous residue in B. stearothermophilusTyrRS (Asp176) has also been implicated as a determinant of amino acid specificity in earlier studies [de Prat Gay, G., Duckworth, H. W., and Fersht, A. R. (1993) FEBS Lett. 318, 167-171]. This striking similarity in the function of a highly conserved residue found in both TrpRS and TyrRS provides mechanistic support for a common origin of the two enzymes.     

Cysteine residues at the active site of glutamine synthetase from spinach leaves

Titration of cysteine residues of spinach glutamine synthetase with 5-5' dithiobis (2-nitrobenzoic acid) indicates that there are five such residues per monomer of enzyme and that two of these five are on the surface of the molecule. The presence of substrates, or either of the competitive inhibitors methionine sulfoximine or phosphinothricin, completely protects both of the surface sulfhydryls from titration. This suggests that both are located at the active site. In the absence of Mg2+ and ATP, both surface sulfhydryls must be modified before loss of activity. We conclude that while both of the cysteine residues are located at the active site, only one of them may be involved in catalysis. Because the cysteine residue which is implicated in catalysis can be protected by Mg2+ and ATP, we believe that it may be located at or near the binding site of these ligands.     

tRNA-dependent active site assembly in a class I aminoacyl-tRNA synthetase

The crystal structure of ligand-free E. coli glutaminyl-tRNA synthetase (GlnRS) at 2.4 A resolution shows that substrate binding is essential to construction of a catalytically proficient active site. tRNA binding generates structural changes throughout the enzyme, repositioning key active site peptides that bind glutamine and ATP. The structure gives insight into longstanding questions regarding the tRNA dependence of glutaminyl adenylate formation, the coupling of amino acid and tRNA selectivities, and the roles of specific pathways for transmission of tRNA binding signals to the active site. Comparative analysis of the unliganded and tRNA-bound structures shows, in detail, how flexibility is built into the enzyme architecture and suggests that the induced-fit transitions are a key underlying determinant of both amino acid and tRNA specificity.     

The conformations of a substrate and a product bound to the active site of S-adenosylmethionine synthetase

S-Adenosylmethionine (AdoMet) is the most widely used alkyl group donor in biological systems. The formation of AdoMet from ATP and L-methionine is catalyzed by S-adenosylmethionine synthetase (AdoMet synthetase). Elucidation of the conformations of enzyme-bound substrates, product, and inhibitors is important for the understanding of the catalytic mechanism of the enzyme and the design of new inhibitors. To obtain structural data for enzyme-bound substrates and product, we have used two-dimensional transferred nuclear Overhauser effect spectroscopy to determine the conformation of enzyme-bound AdoMet and 5'-adenylyl imidodiphosphate (AMPPNP). AMPPNP, an analogue of ATP, is resistant to the ATP hydrolysis activity of AdoMet synthetase because of the presence of a nonhydrolyzable NH-link between the beta- and gamma-phosphates but is a substrate for AdoMet formation during which tripolyphosphate is produced. AdoMet and AMPPNP both bind in an anti conformation about the glycosidic bond. The ribose rings are in C3'-exo and C4'-exo conformations in AdoMet and AMPPNP, respectively. The differences in ribose ring conformations presumably reflect the different steric requirements of the C5' substituents in AMPPNP and AdoMet. The NMR-determined conformations of AdoMet and AMPPNP were docked into the E. coli AdoMet synthetase active site taken from the enzyme.ADP. Pi crystal structure. Since there are no nonexchangeable protons either in the carboxy-terminal end of the methionine segment of AdoMet or in the tripolyphosphate segment of AMPPNP, these portions of the molecules were modeled into the enzyme active site. The interactions of AdoMet and AMPPNP with the enzyme predict the location of the methionine binding site and suggest how the positive charge formed on the sulfur during AdoMet synthesis is stabilized.     

Evidence for a second histidine at the active site of succinyl-CoA synthetase from Escherichia coli.

Ethoxyformic anhydride was used to demonstrate the existence of a second important histidine in succinyl-CoA synthetase from Escherichia coli. Differential labeling of the enzyme by [3H]ethoxyformic anhydride gave a stoichiometry of one important histidine per alpha beta catalytic unit. Data are presented suggesting that this residue and an important thiol group on the beta subunit (Collier, G., and Nishimura, J.S. (1978) J. Biol. Chem. 253, 4938-4943) interact with each other during catalysis. A mechanism of action involving these 2 residues is proposed for one of the partial reactions catalyzed by succinyl-CoA synthetase.     

Oxidation of the active site of glutamine synthetase: conversion of arginine-344 to gamma-glutamyl semialdehyde.

Metal-catalyzed oxidative modification of proteins is implicated in a number of physiologic and pathologic processes. The reaction is presumed to proceed via a site-specific free radical mechanism, with the site-specificity conferred by a cation-binding site on the protein. The oxidation of bacterial glutamine synthetase has been studied in detail, providing the opportunity to examine whether the oxidation is consistent with a site-specific radical reaction. Oxidation leads to the appearance of carbonyl groups in amino acid side chains of the protein, and labeling of those carbonyl groups with fluorescein-amine facilitated purification of the oxidized peptide from a tryptic digest. The oxidized residue was arginine-344, which was converted to a gamma-glutamyl semialdehyde residue. Histidine-269 had previously been shown to be converted to asparagine during metal-catalyzed oxidation. Both arginine-344 and histidine-269 are situated at the metal-nucleotide binding pocket of the enzyme's active site, thus establishing the site-specificity of the oxidation.