Mammalian folylpoly-gamma-glutamate synthetase. 3. Specificity for folate analogues

A variety of folate analogues were synthesized to explore the specificity of the folate binding site of hog liver folylpolyglutamate synthetase and the requirements for catalysis. Modifications of the internal and terminal glutamate moieties of folate cause large drops in on rates and/or affinity for the protein. The only exceptions are glutamine, homocysteate, and ornithine analogues, indicating a less stringent specificity around the delta-carbon of glutamate. It is proposed that initial folate binding to the enzyme involves low-affinity interactions at a pterin and a glutamate site and that the first glutamate bound is the internal residue adjacent to the benzoyl group. Processive movement of the polyglutamate chain through the glutamate site and a possible conformational change in the protein when the terminal residue is bound would result in tight binding and would position the gamma-carboxyl of the terminal glutamate in the correct position for catalysis. Steric limitations imposed on the internal glutamate residues that loop out and additional steric constraints imposed by binding of different pterin moieties would be expected to effect slight conformational changes in the protein and/or the terminal glutamate and would explain the decrease in on rate and catalytic rate with increased polyglutamate chain length, and the differential effect of one-carbon substitution on the catalytic rate with polyglutamate derivatives. The 4-amino substitution of folate increases the on rate for monoglutamate derivatives but severely impairs catalysis with diglutamate derivatives. Pteroylornithine derivatives are the first potent and specific inhibitors of folylpolyglutamate synthetase to be identified and may act as analogues of reaction intermediates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Crystal structure of the S-adenosylmethionine synthetase ternary complex: a novel catalytic mechanism of S-adenosylmethionine synthesis from ATP and Met

S-Adenosylmethionine synthetase (MAT) catalyzes formation of S-adenosylmethionine (SAM) from ATP and l-methionine (Met) and hydrolysis of tripolyphosphate to PP(i) and P(i). Escherichia coli MAT (eMAT) has been crystallized with the ATP analogue AMPPNP and Met, and the crystal structure has been determined at 2.5 A resolution. eMAT is a dimer of dimers and has a 222 symmetry. Each active site contains the products SAM and PPNP. A modeling study indicates that the substrates (AMPPNP and Met) can bind at the same sites as the products, and only a small conformation change of the ribose ring is needed for conversion of the substrates to the products. On the basis of the ternary complex structure and a modeling study, a novel catalytic mechanism of SAM formation is proposed. In the mechanism, neutral His14 acts as an acid to cleave the C5'-O5' bond of ATP while simultaneously a change in the ribose ring conformation from C4'-exo to C3'-endo occurs, and the S of Met makes a nucleophilic attack on the C5' to form SAM. All essential amino acid residues for substrate binding found in eMAT are conserved in the rat liver enzyme, indicating that the bacterial and mammalian enzymes have the same catalytic mechanism. However, a catalytic mechanism proposed recently by González et al. based on the structures of three ternary complexes of rat liver MAT [González, B., Pajares, M. A., Hermoso, J. A., Guillerm, D., Guillerm, G., and Sanz-Aparicio. J. (2003) J. Mol. Biol. 331, 407] is substantially different from our mechanism.     

Structural studies of inhibition of S-adenosylmethionine synthetase by slow, tight-binding intermediate and product analogues

S-Adenosylmethionine synthetase (ATP: L-methionine S-adenosyltransferase) catalyzes a two-step reaction in which tripolyphosphate (PPPi) is a tightly bound intermediate. Diimidotriphosphate (O(3)P-NH-PO(2)-NH-PO(3); PNPNP), a non-hydrolyzable analogue of PPPi, is the most potent known inhibitor of AdoMet synthetase with a K(i) of 2 nM. The structural basis for the slow, tight-binding inhibition by PNPNP has been investigated by spectroscopic methods. UV difference spectra reveal environmental alterations of aromatic protein residues upon PNPNP binding to form the enzyme.2Mg(2+).PNPNP complex, and more extensive changes upon formation of the enzyme.2Mg(2+).PNPNP.AdoMet complex. Stopped-flow kinetic studies of complex formation revealed that two slow isomerizations follow PNPNP binding in the presence of AdoMet, in contrast to the lower affinity, rapid-equilibrium binding in the absence of AdoMet. (31)P NMR spectra of enzyme complexes with PNPNP revealed electronic perturbations of each phosphorus atom by distinct upfield chemical shifts for each of the three phosphoryl groups in the enzyme.2Mg(2+).PNPNP complex, and further upfield shifts of at least 2 resonances in the complex with AdoMet. Comparison of the chemical shifts for the enzyme-bound PNPNP with the enzyme complexes containing either the product analogue O(3)P-NH-PO(3) or O(3)P-O-PO(2)-NH-PO(3) indicates that the shifts on binding are largest at the binding sites corresponding to those for the alpha and gamma phosphoryl groups of the nucleotide (-3.1 to -4.1 ppm), while the resonance at the beta phosphoryl group position shifts by -2.1 ppm. EPR spectra of Mn(2+) complexes demonstrate spin coupling between the two Mn(2+) in both enzyme.2Mn(2+).PNPNP and enzyme.2Mn(2+).PNPNP.AdoMet, indicating that the metal ions have comparable distances in both cases. The combined results indicate that formation of the highest affinity complex is associated with protein side chain rearrangements and increased electron density at the ligand phosphorus atoms, likely due to ionization of an -NH- group of the inhibitor. The energetic feasibility of ionization of a -NH- group when two Mg(2+) ions are bound to O(3)P-NH-PO(3) is supported by density functional theoretical calculations on model chelates. This mode of interaction is uniquely available to compounds with P-NH-P linkages and may be possible with other proteins in which multiple cations coordinate a polyphosphate chain.     

Slow binding inhibition of S-adenosylmethionine synthetase by imidophosphate analogues of an intermediate and product.

S-Adenosylmethionine (AdoMet) synthetase catalyzes the only known route of biosynthesis of the primary in vivo alkylating agent. Inhibitors of this enzyme could provide useful modifiers of biological methylation and polyamine biosynthetic processes. The AdoMet synthetase catalyzed reaction converts ATP and L-methionine to AdoMet, PP(i), and P(i), with formation of tripolyphosphate as a tightly bound intermediate. This work describes a nonhydrolyzable analogue of the tripolyphosphate (PPP(i)) reaction intermediate, diimidotriphosphate (O(3)P-NH-PO(2)-NH-PO(3)(5)(-)), as a potent inhibitor. In the presence of AdoMet, PNPNP is a slow-binding inhibitor with an overall inhibition constant (K(i)) of 2 nM and a dissociation rate of 0.6 h(-)(1). In contrast, in the absence of AdoMet PNPNP is a classical competitive inhibitor with a K(i) of 0.5 microM, a slightly higher affinity than PPP(i) itself (K(i) = 3 microM). The imido analogue of the product pyrophosphate, imidodiphosphate (O(3)P-NH-PO(3)(4)(-)) also displays slow onset inhibition only in the presence of AdoMet, with a K(i) of 0.8 microM, compared to K(i) of 250 microM for PP(i). Circular dichroism spectra of the unliganded enzyme and various complexes are indistinguishable indicating that the protein secondary structure is not greatly altered upon complex formation, suggesting local rearrangements at the active site during the slow binding process. A model based on ionization of the bridging -NH- moiety is presented which could account for the potent inhibition by PNP and PNPNP.     

Metal ion dependence of phosphorothioate ATP analogues in the Bacillus stearothermophilus tyrosyl-tRNA synthetase reaction

Pre-steady-state kinetic analyses on the formation of tyrosyl adenylate from tyrosine and each of the four diastereomers of alpha- and beta-phosphorothioate adenosine triphosphates [ATP alpha S and ATP beta S; Eckstein, F., & Goody, R. (1976) Biochemistry 15, 1685-1691; Yee, D., Armstrong, V. W., & Eckstein, F. (1979) Biochemistry 18, 4116-4123] were performed in the presence of Mg2+, Co2+, and Cd2+ as the divalent metal ion cofactor. A modest preference of 5.5-fold in kappa 3/KA' (where kappa 3 is the rate constant for tyrosyl adenylate formation and KA' is the dissociation constant for ATP, or phosphorothioate ATP, from the E.Tyr.metal.ATP complex) for the Sp ATP alpha S diastereomer and the absence of an inversion of preference when the metal ion is changed suggest that there is a stereospecific enzyme-alpha-phosphate interaction and that there is no direct metal ion interaction with the alpha-phosphate. The extent of reaction of the ATP alpha S diastereomers (30-50%) implies that these analogues are more susceptible to the hydrolytic site reaction previously reported for this enzyme [Wells, T. N. C., & Fersht, A. R. (1986) Biochemistry 25, 1881-1886]. The strong preference in kappa 3/KA' for the RP ATP beta S diastereomer (16-fold for Mg2+ and 50-fold for Co2+) is indicative of a stereospecific interaction with the pro SP beta oxygen of ATP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phytohormonal regulation of S-adenosylmethionine synthetase and S-adenosylmethionine levels in dwarf pea epicotyls.

A significant stimulation (2- to 2.5-fold) of AdoMet synthetase was witnessed in glibberellicd acid (GA3, 1 microM)-treated epicotyls of the dwarf pea (Pisum sativum). This was accompanied by a 2.4-fold increase in the endogenous pool of S-adenosylmethionine. Both abscisic acid (10 microM) and cycloheximide (20 micrograms/ml) inhibited the GA3-mediated enhancement of AdoMet synthetase activity. Three isozymes of AdoMet synthetase were detected in GA3-treated epicotyls, whereas a single activity peak was observed in controls. Thus, GA3 seems to control the induction of two new isozymes of AdoMet synthetase in the dwarf pea. By contrast, the tall pea exhibited three isozymes of AdoMet synthetase even in the absence of GA3 treatment. High concentration of L-methionine (2 mM) mimicked the GA3-elicited induction of two new isozymes of AdoMet synthetase in dwarf pea epicotyls.     

Conserved positions for ribose recognition: importance of water bridging interactions among ATP,ADP and FAD-protein complexes

Analysis of the spatial arrangement of protein and water atoms that form polar interactions with ribose has been performed for a structurally non-redundant dataset of ATP, ADP and FAD-protein complexes. The 26 ligand-protein structures were separated into two groups corresponding to the most populated furanose ring conformations (N and S-domains). Four conserved positions were found for S-domain protein-ligand complexes and five for N-domain complexes. Multiple protein folds and secondary structural elements were represented at a single conserved position. The following novel points were revealed: (i) Two complementary positions sometimes combine to describe a putative atomic spatial location for a specific conserved binding spot. (ii) More than one third of the interactions scored were water-mediated. Thus, conserved spatial positions rich in water atoms are a significant feature of ribose-protein complexes.     

Specificity and genetics of S-adenosylmethionine transport in Saccharomyces cerevisiae.

The specificity of a transport system for S-adenosylmethionine was determined through the use of structurally related derivatives. Of the compounds tested, the analogues S-adenosylethionine and S-inosylmethionine and the naturally occurring compounds S-adenosyl-(5')-3-methylthiopropylamine and S-adenosylhomocysteine competitively inhibited uptake of the sulfonium compound. Ki values for these compounds indicate that the order of affinity for the transport protein is S-adenosylmethionine congruent to S-adenosyl-(5')-3-methyl-thiopropylamine greater than S-adenosylethionine greater than S-inosylmethionine greater than S-adenosylhomocysteins. S-adenosyl-(2-hydroxy-4-methylthio)butyric acid exerted inhibition of a mixed type. S-insoyl-(2-hydroxy-4-methylthio)butyric acid, S-inosylhomocysteine, and S-ribosylhomocysteine were without effect. On the basis of the inhibition data, the methionine-amino, adenine-amino, and methyl groups were identified as group important in the binding of S-adenosylmethionine to the transport protein. Comparison is made with the specificities of various transmethylating enzymes utilizing S-adenosylmethionine. In addition, a number of conventional and temperature-sensitive S-adenosylmethionine transport mutants were isolated and analyzed in an attempt to identify the structural character of the specific transport protein(s). The data obtained suggest that only a single gene (a single polypeptide) is involved in specific S-adenosylmethionine transport. Apparent interallelic complementation supports the assumption that the functional form of the protein is composed of two or more copies of a monomer.     

Identification of the reactive sulfhydryl groups of S-adenosylmethionine synthetase

S-Adenosylmethionine synthetase from Escherichia coli is rapidly inactivated by N-ethylmaleimide. In the presence of excess N-ethylmaleimide inactivation follows pseudo first-order kinetics, and loss of enzyme activity correlates with the incorporation of 2 eq of N-[ethyl-2-3H]maleimide/subunit. Preincubation of the enzyme with methionine and the ATP analog adenylylimidodiphosphate reduced the rate of N-ethylmaleimide incorporation more than 30-fold. Two N-[ethyl-2-3H]maleimide-labeled tryptic peptides were purified from the modified enzyme by reverse phase high performance liquid chromatography. The modified residues were identified as cysteine 90 and cysteine 240 by comparison of the amino acid compositions of these peptides with the protein sequence. These are the first residues to be implicated in the activity and/or structure of the enzyme. N-Ethylmaleimide-modified S-adenosylmethionine synthetase exists mainly as a dimer in conditions where the native enzyme is a tetramer. Accumulation of the dimer parallels the loss of the enzyme activity. When an enzyme sample was partially inactivated, separation of tetrameric and dimeric enzyme forms by gel filtration revealed that the residual enzyme activity was solely present in the tetramer and N-[ethyl-2-3H] maleimide was present predominantly in the dimer. Gel filtration studies of the tetramer-dimer equilibrium for the native enzyme indicated that the dissociation constant between the tetramer and dimers is less than 6 x 10(-11) M. Similar studies for the N-ethylmaleimide-modified protein indicated that the dissociation constant of the tetramer is approximately 4 x 10(-4) M. Upon modification the strength of dimer-dimer interactions is diminished by at least 9 kcal/mol.     

Syntheses of stable,synthetic diadenosine polyphosphate analogues using recombinant histidine-tagged lysyl tRNA synthetase (LysU)

Recombinant Escherichia coli lysyl-tRNA synthase (LysU) has been previously utilised in the production of stabile, synthetic diadenosine polyphosphate (Ap_nA) analogues. Here we report on the extended use of a new recombinant histidine residue-tagged LysU as a tool for highly controlled phosphatephosphate bond formation between nucleotides, avoiding the need for complex protecting group chemistries. Resulting high yielding tandem LysU-based biosynthetic–synthetic/synthetic–biosynthetic strategies emerge for the preparation of varieties of Ap_nA analogues directly from inexpensive natural nucleotides and nucleosides. Analogues so formed make a useful small library with which to probe Ap_nA activities in vitro and in vivo leading to the discovery of new, potentially potent biopharmaceuticals active against chronic pain and other chronic, high-burden disease states.     

Defective transport in S-adenosylmethionine synthetase mutants of Escherichia coli

The transport of several metabolites is decreased in mutant strains of Escherichia coli (Met K, E4 and E40), which contain decreased levels of S-adenosylmethionine synthetase. The rates and extents of uptake for lysine, leucine, methionine, and α-methylglucoside in both whole cells and membrane vesicles isolated from these mutants are 2- to 10-fold lower than in corresponding preparations from wild-type cells, although proline uptake is normal. The addition of S-adenosylmethionine to cultures of strain E40 can partially restore the rate and extent of lysine uptake. Lysine transport is lower in mutant vesicles in the presence of either d-lactate, succinate, α-hydroxylbutyrate, or NADH even though these substrates are oxidized at rates comparable to those in wild-type vesicles. This suggests that the defect is not related to the ability of vesicles to oxidize electron donors, but is very likely related to the ability of mutant vesicles to couple respiration to lysine transport. In addition, temperature-induced efflux of α-methylglucoside phosphate and dinitrophenol-induced efflux of lysine are similar in both the mutant and wild-type membranes, indicating that the barrier properties of the membrane and the activity of the lysine carrier are normal.     

The sequence of metK, the structural gene for S-adenosylmethionine synthetase in Escherichia coli

The DNA sequence of the Escherichia coli metK gene has been determined. Protein sequence data for purified S-adenosylmethionine synthetase have also been obtained and confirm that metK is the structural gene for S-adenosylmethionine synthetase in E. coli. The sequence of the amino-terminal 35 residues of purified S-adenosylmethionine synthetase localizes the beginning of the coding region of the DNA. The open reading frame extends 1152 bases and codes for a 384-residue protein of Mr = 41,941. The gene is transcribed clockwise on the E. coli chromosome. The DNA region 5' to the coding region was found to contain symmetrical sequences suggestive of operator structures and homologous to sequences upstream from other met genes sharing the same regulatory mechanism.     

Enzymatic properties of S-adenosylmethionine synthetase from the archaeon Methanococcus jannaschii

S-Adenosylmethionine synthetase (ATP:l-methionine S-adenosyltransferase, MAT) catalyzes a unique enzymatic reaction that leads to formation of the primary biological alkylating agent. MAT from the hyperthermophilic archaeon Methanococcus jannaschii (MjMAT) is a prototype of the newly discovered archaeal class of MAT proteins that are nearly unrecognizable in sequence when compared with the class that encompasses both the eucaryal and bacterial enzymes. In this study the functional properties of purified recombinant MjMAT have been evaluated. The products of the reaction are AdoMet, PP(i), and P(i); >90% of the P(i) originates from the gamma-phosphoryl group of ATP. The circular dichroism spectrum of the dimeric MjMAT indicates that the secondary structure is more helical than the Escherichia coli counterpart (EcMAT), suggesting a different protein topology. The steady state kinetic mechanism is sequential, with random addition of ATP and methionine; AdoMet is the first product released, followed by release of PP(i) and P(i). The substrate specificity differs remarkably from the previously characterized MATs; the nucleotide binding site has a very broad tolerance of alterations in the adenosine moiety. MjMAT has activity at 70 degrees C comparable with that of EcMAT at 37 degrees C, consistent with the higher temperature habitat of M. jannaschii. The activation energy for AdoMet formation is larger than that for the E. coli MAT-catalyzed reaction, in accord with the notion that enzymes from thermophilic organisms are often more rigid than their mesophilic counterparts. The broad substrate tolerance of this enzyme proffers routes to preparation of novel AdoMet analogs.