inosinic-acid and hadacidin

In enzymes that conduct complex reactions involving several substrates and chemical transformations, the active site must reorganize at each step to complement the transition state of that chemical step. Adenylosuccinate synthetase (ADSS) utilizes a molecule each of guanosine 5'-monophosphate (GTP) and aspartate to convert inosine 5'-monophosphate (IMP) into succinyl adenosine 5'-monophosphate (sAMP) through several kinetic intermediates. Here we followed catalysis by ADSS through high-resolution vibrational spectral fingerprints of each substrate and intermediate involved in the forward reaction. Vibrational spectra show differential ligand distortion at each step of catalysis, and band positions of substrates are influenced by binding of cosubstrates. We found that the bound IMP is distorted toward its N1-deprotonated form even in the absence of any other ligands. Several specific interactions between GTP and active-site amino acid residues result in large Raman shifts and contribute substantially to intrinsic binding energy. When both IMP and GTP are simultaneously bound to ADSS, IMP is converted into an intermediate 6-phosphoryl inosine 5'-monophosphate (6-pIMP). The 6-pIMP·ADSS complex was found to be stable upon binding of the third ligand, hadacidin (HDA), an analogue of l-aspartate. We find that in the absence of HDA, 6-pIMP is quickly released from ADSS, is unstable in solution, and converts back into IMP. HDA allosterically stabilizes ADSS through local conformational rearrangements. We captured this complex and determined the spectra and structure of 6-pIMP in its enzyme-bound state. These results provide important insights into the exquisite tuning of active-site interactions with changing substrate at each kinetic step of catalysis.

Topics: Adenosine Monophosphate; Adenylosuccinate Synthase; Aspartic Acid; Binding Sites; Catalysis; Catalytic Domain; Crystallography, X-Ray; Glycine; Guanosine Triphosphate; Inosine Monophosphate; Kinetics; Ligands; Methanocaldococcus; Models, Molecular; Protein Conformation

In the absence of the de novo purine nucleotide biosynthetic pathway in parasitic protozoa, purine salvage is of primary importance for parasite survival. Enzymes of the salvage pathway are, therefore, good targets for anti-parasitic drugs. Adenylosuccinate synthetase (AdSS), catalysing the first committed step in the synthesis of AMP from IMP, is a potential target for anti-protozoal chemotherapy. We report here the crystal structure of adenylosuccinate synthetase from the malaria parasite, Plasmodium falciparum, complexed to 6-phosphoryl IMP, GDP, Mg2+ and the aspartate analogue, hadacidin at 2 A resolution. The overall architecture of P. falciparum AdSS (PfAdSS) is similar to the known structures from Escherichia coli, mouse and plants. Differences in substrate interactions seen in this structure provide a plausible explanation for the kinetic differences between PfAdSS and the enzyme from other species. Additional hydrogen bonding interactions of the protein with GDP may account for the ordered binding of substrates to the enzyme. The dimer interface of PfAdSS is also different, with a pronounced excess of positively charged residues. Differences highlighted here provide a basis for the design of species-specific inhibitors of the enzyme.

Topics: Adenylosuccinate Synthase; Amino Acid Sequence; Animals; Binding Sites; Crystallography, X-Ray; Dimerization; Glycine; Guanosine Diphosphate; Hydrogen Bonding; Inosine Monophosphate; Magnesium; Models, Molecular; Molecular Sequence Data; Molecular Structure; Plasmodium falciparum; Protein Conformation; Recombinant Proteins; Sequence Homology, Amino Acid

A complete set of substrate/substrate analogs of adenylosuccinate synthetase from Escherichia coli induces dimer formation and a transition from a disordered to an ordered active site. The most striking of the ligand-induced effects is the movement of loop 40-53 by up to 9 A. Crystal structures of the partially ligated synthetase, which either combine IMP and hadacidin or IMP, hadacidin, and Mg(2+)-pyrophosphate, have ordered active sites, comparable with the fully ligated enzyme. More significantly, a crystal structure of the synthetase with IMP alone exhibits a largely ordered active site, which includes the 9 A movement of loop 40-53 but does not include conformational adjustments to backbone carbonyl 40 (Mg(2+) interaction element) and loop 298-304 (L-aspartate binding element). Interactions involving the 5'-phosphoryl group of IMP evidently trigger the formation of salt links some 30 A away. The above provides a structural basis for ligand binding synergism, effects on k(cat) due to mutations far from the site of catalysis, and the complete loss of substrate efficacy due to minor alterations of the 5'-phosphoryl group of IMP.

Topics: Adenylosuccinate Synthase; Amino Acid Sequence; Binding Sites; Crystallography, X-Ray; Escherichia coli; Glycine; Guanosine Triphosphate; Inosine Monophosphate; Models, Molecular; Protein Conformation

Prokaryotes have a single form of adenylosuccinate synthetase that controls the committed step of AMP biosynthesis, but vertebrates have two isozymes of the synthetase. The basic isozyme, which predominates in muscle, participates in the purine nucleotide cycle, has an active site conformation different from that of the Escherichia coli enzyme, and exhibits significant differences in ligand recognition. Crystalline complexes presented here of the recombinant basic isozyme from mouse show the following. GTP alone binds to the active site without inducing a conformational change. IMP in combination with an acetate anion induces major conformational changes and organizes the active site for catalysis. IMP, in the absence of GTP, binds to the GTP pocket of the synthetase. The combination of GTP and IMP results in the formation of a stable complex of 6-phosphoryl-IMP and GDP in the presence or absence of hadacidin. The response of the basic isozyme to GTP alone differs from that of synthetases from plants, and yet the conformation of the mouse basic and E. coli synthetases in their complexes with GDP, 6-phosphoryl-IMP, and hadacidin are nearly identical. Hence, reported differences in ligand recognition among synthetases probably arise from conformational variations observed in partially ligated enzymes.

Topics: Adenylosuccinate Synthase; Animals; Binding Sites; Catalysis; Glycine; Guanosine Diphosphate; Guanosine Triphosphate; Hydrogen Bonding; Inosine Monophosphate; Ligands; Mice; Models, Molecular; Muscles; Protein Conformation; Recombinant Proteins

Topics: Adenine Nucleotides; Adenylosuccinate Synthase; Anaerobiosis; Animals; Aspartic Acid; Carcinoma, Ehrlich Tumor; Cell Hypoxia; Glutamine; Glycine; Inosine; Inosine Monophosphate; Mice; Oxidation-Reduction; Oxidative Stress; Oxygen

The site of action of hydantocidin was probed using Arabidopsis thaliana plants growing on agar plates. Herbicidal effects were reversed when the agar medium was supplemented with AMP, but not IMP or GMP, suggesting that hydantocidin blocked the two-step conversion of IMP to AMP in the de novo purine biosynthesis pathway. Hydantocidin itself did not inhibit adenylosuccinate synthetase or adenylosuccinate lyase isolated from Zea mays. However, a phosphorylated derivative of hydantocidin, N-acetyl-5'-phosphohydantocidin, was a potent inhibitor of the synthetase but not of the lyase. These results identify the site of action of hydantocidin and establish adenylosuccinate synthetase as an herbicide target of commercial potential.

Topics: Adenosine Monophosphate; Adenylosuccinate Lyase; Adenylosuccinate Synthase; Antidotes; Arabidopsis; Enzyme Inhibitors; Glycine; Herbicides; Hydantoins; Inosine Monophosphate; Pentosephosphates; Zea mays

Inosine 5'-monophosphate (IMP) reamination in skeletal muscle fiber sections of the rat hindlimb was studied. High IMP concentrations were established during ischemic contractions in each fiber section: 3.1, 2.8, or 0.6 mumol/g in the fast-twitch white (FTW), fast-twitch red (FTR), and slow-twitch red (STR) muscle sections, respectively. Thereafter blood flow was restored and stimulation was discontinued to allow reamination of IMP. After 0, 2, 5, 10, 15, or 20 min of recovery, muscle sections were freeze-clamped and analyzed for metabolite contents. IMP was nearly fully reaminated after 10 and 20 min of recovery in STR and FTR muscles, respectively. Reamination in TW fibers was delayed and slower, with only 50% of the IMP reaminated after 20 min of recovery. Significant recovery (approximately 75%) of phosphocreatine occurs in each fiber section before the onset of reamination. Reamination was also evaluated after high-speed treadmill running with or without inhibition of reamination by hadacidin. Running resulted in large accumulations of IMP in FTW and FTR fibers (3.5 and 1.4 mumul/g, respectively); IMP in FTR fibers was higher with hadacidin treatment. Reamination after running was much greater in FTR than in FTW fibers and was associated with recovery of phosphocreatine. After running, the purine degradation products inosine and hypoxanthine were increased in FTW and FTR fibers in normal and hadacidin-treated animals. Plasma inosine, hypoxanthine, and urate increased after exercise; concentrations continued to increase if reamination was inhibited by hadacidin. These results demonstrate that when muscle IMP is increased, subsequent degradation and loss of purines occur. Rapid reamination should minimize the quantity of purine lost from muscle and limit the metabolic cost of replenishing purines by the de novo synthesis or salvage pathways.

Topics: Adenosine Monophosphate; Amines; Animals; Blood; Electric Stimulation; Glycine; Inosine Monophosphate; Male; Motor Activity; Muscle, Skeletal; Rats; Rats, Sprague-Dawley

Crystal structures of adenylosuccinate synthetase from Esherichia coli complexed with Mg2+, IMP, GDP, NO3- and hadacidin at 298 and 100 K have been refined to R-factors of 0.188 and 0.206 against data to 2.8 A and 2.5 A resolution, respectively. Conformational changes of up to 9 A relative to the unligated enzyme occur in loops that bind to Mg2+, GDP, IMP and hadacidin. Mg2+ binds directly to GDP, NO3-, hadacidin and the protein, but is only five-coordinated. Asp13, which approaches, but does not occupy the sixth coordination site of Mg2+, hydrogen bonds to N1 of IMP. The nitrogen atom of NO3- is approximately 2.7 A from O6 of IMP, reflecting a strong electrostatic interaction between the electron-deficient nitrogen atom and the electron-rich O6. The spatial relationships between GDP, NO3- and Mg2+ suggest an interaction between the beta,gamma-bridging oxygen atom of GTP and Mg2+ in the enzyme-substrate complex. His41 hydrogen bonds to the beta-phosphate group of GDP and approaches bound NO3-. The aldehyde group of hadacidin coordinates to the Mg2+, while its carboxyl group interacts with backbone amide groups 299 to 303 and the side-chain of Arg303. The 5'-phosphate group of IMP interacts with Asn38, Thr129, Thr239 and Arg143 (from a monomer related by 2-fold symmetry). A mechanism is proposed for the two-step reaction governed by the synthetase, in which His41 and Asp13 are essential catalytic side-chains.

Topics: Adenylosuccinate Synthase; Binding Sites; Crystallography, X-Ray; Electrochemistry; Escherichia coli; Glycine; Guanosine Diphosphate; Inosine Monophosphate; Magnesium; Models, Molecular; Molecular Sequence Data; Molecular Structure; Nitrates; Protein Conformation

De novo synthesis precursors of the purine second messengers adenosine, guanosine and inosine are adenosine, guanosine and inosine monophosphate (AMP, GMP, IMP), respectively. Inhibitors of the de novo purinergic synthesis pathways for AMP, GMP and IMP by hadacidin, mycophenolic acid and azaserine, respectively, or adenosine, guanosine or inosine alone or in combination were given every 4 or 6 hours in vivo. Treatments were given into the ovarian vascular pedicle sheath adjacent to the luteal-bearing ovary in three separate experiments to determine whether purines were involved in development of the corpus luteum. Hadacidin lowered AMP (p < or = 0.01) and azaserine tended to lower IMP and the GMP: AMP ratio (p < or = 01) while mycophenolic acid tended to lower the GMP:AMP ratio (p < or = 0.1) in luteal tissue. Azaserine (150 mg) increased progesterone (p < or = 0.01) on some days but guanosine or inosine had no effect on profiles of progesterone in jugular blood of the developing corpus luteum (p > or = 0.1). Azaserine (500 micrograms) tended to lower progesterone in jugular blood (p < or = 0.1) while profiles of progesterone did not differ among guanosine or inosine or adenosine, guanosine and inosine plus hadacidin, mycophenolic acid and azaserine treatment groups compared to controls (p > or = 0.1). Weights of corpora lutea or composition of cell types in the corpus luteum or their viability were not affected by adenosine, guanosine, inosine, hadacidin, mycophenolic acid or azaserine (p > or = 0.1). Since profiles of jugular progesterone did not differ between treatments during development of the corpus luteum, these results suggest that progesterone production by the developing corpus luteum is a) less dependent on de novo synthesized purines or b) there may be a non-purinergic-dependent second messenger system controlling biosynthesis of steroids in the developing ovine corpus luteum.

Topics: Adenosine Monophosphate; Animals; Antibiotics, Antineoplastic; Azaserine; Chromatography, High Pressure Liquid; Corpus Luteum; Estrus; Female; Glycine; Guanosine Monophosphate; Inosine Monophosphate; Mycophenolic Acid; Pregnancy; Progesterone; Purines; Radioimmunoassay; Sheep

The extent of purine nucleotide cycle (PNC) turnover [i.e., the cyclic deamination of adenosine 5'-monophosphate (AMP) and reamination of inosine 5'-monophosphate (IMP)] was estimated in fast-twitch muscle of pentobarbital sodium anesthetized rats during in situ stimulation, by the accumulation of excess IMP after blocking IMP reamination with the adenylosuccinate synthetase inhibitor, hadacidin. Sodium hadacidin (100 mg/kg, iv) did not alter IMP production and was effective in blocking IMP reamination by 80%. Hadacidin had no effect on IMP levels in muscle at rest or during mild stimulation (1 Hz for 30 min). During 30 min of more intense stimulation (5 Hz), hadacidin treatment resulted in a threefold increase in IMP in the fast-twitch white gastrocnemius section, but had no effect in the fast-twitch red section. The IMP in the fast-twitch white section did not accumulate over the 30-min period, but was produced initially (within 5 min) and kept for being reaminated by hadacidin. Thus, the IMP formation and IMP reamination evident in the saline-injected animals did not occur concurrently. This suggests that the IMP reamination arm of the PNC is not an important pathway in "working" muscle.

Topics: Adenosine Diphosphate; Adenosine Monophosphate; Adenosine Triphosphate; Ammonia; Animals; Formaldehyde; Glycine; Inosine Monophosphate; Inosine Nucleotides; Lactates; Male; Muscle Contraction; Muscles; Phosphocreatine; Physical Exertion; Rats