RESUMEN
Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) is the key enzyme in purine base salvage in humans and in purine auxotrophs, including Plasmodium falciparum, the leading cause of malaria. Hydrogen/deuterium (H/D) exchange into amide bonds, quantitated by on-line HPLC and mass spectrometry, has been used to compare the dynamic and conformational properties of human HGPRT alone, the HGPRT-GMP-Mg(2+) complex, the HGPRT-IMP-MgPPi <==> HGPRT-Hx-MgPRPP equilibrating mixture, and the transition-state analogue complex HGPRT-ImmGP-MgPPi. The rate and extent of H/D exchange of 26 peptic peptides, spanning 91% of the primary structure, have been monitored. Human HGPRT has 207 amide H/D exchange sites. After 1 h in D2O, HGPRT alone exchanges 160, HGPRT-GMP-Mg(2+) exchanges 154, the equilibrium complex exchanges 139, and the transition-state analogue complex exchanges 126 of these amide protons. H/D exchange rates are correlated with structure for peptides in (1) catalytic site loops, (2) a connected peptide of the subunit interface of the tetramer, and (3) a loop buried in the catalytic site. Structural properties related to H/D exchange are defined from crystallographic studies of the HGPRT-GMP-Mg(2+) and HGPRT-ImmGP-MgPPi complexes. Transition-state analogue binding strengthens the interaction between subunits and tightens the catalytic site loops. The solvent exchange dynamics in specific peptides correlates with hydrogen bond patterns, solvent access, crystallographic B-factors, and ligand exchange rates. Solvent exchange reveals loop dynamics in the free enzyme, Michaelis complexes, and the complex with the bound transition-state analogue. Proton transfer paths, rather than dynamic motion, are required to explain exchange into a buried catalytic site peptide in the complex with the bound transition-state analogue.
Asunto(s)
Inhibidores Enzimáticos/química , Hipoxantina Fosforribosiltransferasa/antagonistas & inhibidores , Hipoxantina Fosforribosiltransferasa/química , Pirimidinonas/química , Pirroles/química , Secuencia de Aminoácidos , Sitios de Unión , Dominio Catalítico , Cromatografía Líquida de Alta Presión , Deuterio/metabolismo , Difosfatos/química , Humanos , Hipoxantina Fosforribosiltransferasa/metabolismo , Isoleucina/química , Leucina/química , Sustancias Macromoleculares , Magnesio/química , Compuestos de Magnesio/química , Espectrometría de Masas , Datos de Secuencia Molecular , Pepsina A/metabolismo , Fragmentos de Péptidos/antagonistas & inhibidores , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Fenilalanina/química , ProtonesRESUMEN
The yeast Sir2 protein, required for transcriptional silencing, has an NAD(+)-dependent histone deacetylase (HDA) activity. Yeast extracts contain a NAD(+)-dependent HDA activity that is eliminated in a yeast strain from which SIR2 and its four homologs have been deleted. This HDA activity is also displayed by purified yeast Sir2p and homologous Archaeal, eubacterial, and human proteins, and depends completely on NAD(+) in all species tested. The yeast NPT1 gene, encoding an important NAD(+) synthesis enzyme, is required for rDNA and telomeric silencing and contributes to silencing of the HM loci. Null mutants in this gene have significantly reduced intracellular NAD(+) concentrations and have phenotypes similar to sir2 null mutants. Surprisingly, yeast from which all five SIR2 homologs have been deleted have relatively normal bulk histone acetylation levels. The evolutionary conservation of this regulated activity suggests that the Sir2 protein family represents a set of effector proteins in an evolutionarily conserved signal transduction pathway that monitors cellular energy and redox states.
Asunto(s)
Proteínas Fúngicas/fisiología , Histona Desacetilasas/fisiología , NAD/fisiología , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae , Transactivadores/fisiología , ADN Ribosómico/genética , Histonas/metabolismo , Filogenia , Poli(ADP-Ribosa) Polimerasas/fisiología , Recombinación Genética , Saccharomyces cerevisiae/enzimología , Sirtuina 1 , Sirtuina 2 , SirtuinasRESUMEN
Giardia lamblia, the protozoan parasite responsible for giardiasis, requires purine salvage from its host for RNA and DNA synthesis. G. lamblia expresses an unusual purine phosphoribosyltransferase with a high specificity for guanine (GPRTase). The enzyme's sequence significantly diverges from those of related enzymes in other organisms. The transition state analogue immucillinGP is a powerful inhibitor of HGXPRTase from malaria [Li, C. M., et al. (1999) Nat. Struct. Biol. 6, 582-587] and is also a 10 nM inhibitor of G. lamblia GPRTase. Cocrystallization of GPRTase with immucillinGP led unexpectedly to a GPRTase.immucillinG binary complex with an open catalytic site loop. Diffusion of ligands into preformed crystals gave a GPRTase.immucillinGP.Mg(2+).pyrophosphate complex in which the open loop is stabilized by crystal contacts. G. lamblia GPRTase exhibits substantial structural differences from known purine phosphoribosyltransferases at positions remote from the catalytic site, but conserves most contacts to the bound inhibitor. The filled catalytic site with an open catalytic loop provides insight into ligand binding. One active site Mg(2+) ion is chelated to pyrophosphate, but the other is chelated to two conserved catalytic site carboxylates, suggesting a role for these amino acids. This arrangement of Mg(2+) and pyrophosphate has not been reported in purine phosphoribosyltransferases. ImmucillinG in the binary complex is anchored by its 9-deazaguanine group, and the iminoribitol is disordered. No Mg(2+) or pyrophosphate is detected; thus, the 5'-phosphoryl group is needed to immobilize the iminoribitol prior to magnesium pyrophosphate binding. Filling the catalytic site involves (1) binding the purine ring, (2) anchoring the 5'-phosphate to fix the ribosyl group, (3) binding the first Mg(2+) to Asp125 and Glu126 carboxyl groups and binding Mg(2+).pyrophosphate, and (4) closing the catalytic site loop and formation of bound (Mg(2+))(2). pyrophosphate prior to catalysis. Guanine specificity is provided by two peptide carbonyl oxygens hydrogen-bonded to the exocyclic amino group and a weak interaction to O6. Transition state formation involves N7 protonation by Asp129 acting as the general acid.
Asunto(s)
Giardia lamblia/enzimología , Hipoxantina Fosforribosiltransferasa/química , Animales , Sitios de Unión , Cristalografía por Rayos X , Dimerización , Inhibidores Enzimáticos/química , Modelos Moleculares , Conformación Molecular , Pirimidinonas/química , Pirroles/química , Proteínas Recombinantes/químicaAsunto(s)
Adenosina Trifosfato/química , Pentosiltransferasa/química , Adenosina Trifosfatasas/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Clonación Molecular , Eritrocitos/enzimología , Histidina/análogos & derivados , Histidina/química , Humanos , Cinética , Datos de Secuencia Molecular , Mononucleótido de Nicotinamida/biosíntesis , Pentosiltransferasa/genética , Conformación Proteica , Salmonella/enzimología , Ciclo del Sustrato , Levaduras/enzimologíaRESUMEN
Malaria is a leading cause of worldwide mortality from infectious disease. Plasmodium falciparum proliferation in human erythrocytes requires purine salvage by hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRTase). The enzyme is a target for the development of novel antimalarials. Design and synthesis of transition-state analogue inhibitors permitted cocrystallization with the malarial enzyme and refinement of the complex to 2.0 A resolution. Catalytic site contacts in the malarial enzyme are similar to those of human hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) despite distinct substrate specificity. The crystal structure of malarial HGXPRTase with bound inhibitor, pyrophosphate, and two Mg(2+) ions reveals features unique to the transition-state analogue complex. Substrate-assisted catalysis occurs by ribooxocarbenium stabilization from the O5' lone pair and a pyrophosphate oxygen. A dissociative reaction coordinate path is implicated in which the primary reaction coordinate motion is the ribosyl C1' in motion between relatively immobile purine base and (Mg)(2)-pyrophosphate. Several short hydrogen bonds form in the complex of the enzyme and inhibitor. The proton NMR spectrum of the transition-state analogue complex of malarial HGXPRTase contains two downfield signals at 14.3 and 15.3 ppm. Despite the structural similarity to the human enzyme, the NMR spectra of the complexes reveal differences in hydrogen bonding between the transition-state analogue complexes of the human and malarial HG(X)PRTases. The X-ray crystal structures and NMR spectra reveal chemical and structural features that suggest a strategy for the design of malaria-specific transition-state inhibitors.
Asunto(s)
Inhibidores Enzimáticos/química , Pentosiltransferasa/antagonistas & inhibidores , Pentosiltransferasa/química , Plasmodium falciparum/enzimología , Pirimidinonas/química , Pirroles/química , Animales , Dominio Catalítico , Cristalografía por Rayos X , Humanos , Sustancias Macromoleculares , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Pliegue de Proteína , Estructura Secundaria de Proteína , Protones , Nucleósidos de PurinaRESUMEN
L-Histidinol dehydrogenase catalyzes the biosynthetic oxidation of L-histidinol to L-histidine with sequential reduction of two molecules of NAD. Previous isotope exchange results had suggested that the oxidation of histidinol to the intermediate histidinaldehyde occurred 2-3-fold more rapidly than overall catalysis. In this work, we present kinetic isotope effects (KIE) studies at pH 9.0 and at pH 6.7 with stereospecifically mono- and dideuterated histidinols. The data at pH 9.0 support minimal participation of the first hydride transfer and substantial participation of the second hydride transfer in the overall rate limitation. Stopped-flow experiments with protiated histidinol revealed a small burst of NADH production with stoichiometry of 0.12 per subunit, and 0.25 per subunit with dideuterated histidinol, indicating that the overall first half-reaction was not significantly faster than the second reaction sequence. Results from kcat and kcat/KM titrations with histidinol, NAD, and the alternative substrate imidazolyl propanediol demonstrated an essential base with pKa values between 7.7 and 8.4. In KIE experiments performed at pH 6.7 or with a coenzyme analogue at pH 9. 0, the first hydride transfer became more rate limiting. Kinetic simulations based on rate constants estimated from this work fit well with a mechanism that includes a relatively fast, and thermodynamically unfavorable, hydride transfer from histidinol and a slower, irreversible second hydride transfer from a histidinaldehyde derivative. Thus, although the chemistry of the first hydride transfer is fast, both partial reactions participate in the overall rate limitation.
Asunto(s)
Oxidorreductasas de Alcohol/química , Oxidorreductasas de Alcohol/metabolismo , Salmonella typhimurium/enzimología , Sitios de Unión , Catálisis , Deuterio , Histidinol/química , Histidinol/metabolismo , Concentración de Iones de Hidrógeno , Imidazoles/química , Imidazoles/metabolismo , Cinética , Espectrofotometría/métodos , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
The dimeric zinc metalloenzyme L-histidinol dehydrogenase (HDH) catalyzes an unusual four-electron oxidation of the amino alcohol histidinol via the histidinaldehyde intermediate to the acid product histidine with the reduction of two molecules of NAD. An essential base, with pKa about 8, is involved in catalysis. Here we report site-directed mutagenesis studies to replace each of the five histidine residues (His-98, His-261, His-326, His-366, and His-418) in Salmonella typhimurium with either asparagine or glutamine. In all cases, the overexpressed enzymes were readily purified and behaved as dimers. Substitution of His-261 and His-326 by asparagine caused about 7000- and 500-fold decreases in kcat, respectively, with little change in KM values. Similar loss of activity was also reported for a H261N mutant Brassica HDH [Nagai, A., and Ohta, D. (1994) J. Biochem. 115, 22-25]. Kinetic isotope effects, pH profiles, substrate rescue, and stopped-flow experiments suggested that His-261 and His-326 are involved in proton transfers during catalysis. Sensitivity to metal ion chelator and decreased affinities for metal ions with substitutions at His-261 and His-418 suggested that these two residues are candidates for zinc ion ligands.
Asunto(s)
Oxidorreductasas de Alcohol/genética , Secuencia Conservada/genética , Histidina/genética , Mutagénesis Sitio-Dirigida , Oxidorreductasas de Alcohol/química , Oxidorreductasas de Alcohol/metabolismo , Asparagina/metabolismo , Sitios de Unión/genética , Catálisis , Cationes Bivalentes , Deuterio , Histidina/fisiología , Histidinol/metabolismo , Imidazoles/metabolismo , Cinética , Ligandos , Oxidación-Reducción , Propanoles/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Salmonella typhimurium/enzimología , Especificidad por Sustrato/genética , Zinc/química , Zinc/metabolismoRESUMEN
The proposed transition state for hypoxanthine-guanine phosphoribosyltransferases (HGPRTs) has been used to design and synthesize powerful inhibitors that contain features of the transition state. The iminoribitols (1S)-1-(9-deazahypoxanthin-9-yl)-1,4-dideoxy-1,4-imino-D-ribitol 5-phosphate (immucillinHP) and (1S)-1-(9-deazaguanin-9-yl)-1,4-dideoxy-1,4-imino-D-ribitol 5-phosphate (immucillinGP) are the most powerful inhibitors yet reported for both human and malarial HGPRTs. Equilibrium binding constants are >1,000-fold tighter than the binding of the nucleotide substrate. The NMR spectrum of malaria HGXPRT in the Michaelis complex reveals downfield hydrogen-bonded protons. The chemical shifts move farther downfield with bound inhibitor. The inhibitors are lead compounds for species-specific antibiotics against parasitic protozoa. The high-resolution crystal structure of human HGPRT with immucillinGP is reported in the companion paper.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Hipoxantina Fosforribosiltransferasa/antagonistas & inhibidores , Plasmodium falciparum/enzimología , Pirimidinonas/metabolismo , Pirroles/metabolismo , Animales , Sitios de Unión/efectos de los fármacos , Catálisis/efectos de los fármacos , Difosfatos/metabolismo , Difosfatos/farmacología , Diseño de Fármacos , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/uso terapéutico , Guanosina Monofosfato/metabolismo , Humanos , Enlace de Hidrógeno , Hipoxantina/metabolismo , Hipoxantina Fosforribosiltransferasa/metabolismo , Inosina Monofosfato/metabolismo , Cinética , Compuestos de Magnesio/metabolismo , Compuestos de Magnesio/farmacología , Resonancia Magnética Nuclear Biomolecular , Fosforribosil Pirofosfato/metabolismo , Fosforilación , Unión Proteica/efectos de los fármacos , Protones , Nucleósidos de Purina , Pirimidinonas/farmacología , Pirimidinonas/uso terapéutico , Pirroles/farmacología , Pirroles/uso terapéuticoRESUMEN
The structure of human HGPRT bound to the transition-state analog immucillinGP and Mg2+-pyrophosphate has been determined to 2.0 A resolution. ImmucillinGP was designed as a stable analog with the stereoelectronic features of the transition state. Bound inhibitor at the catalytic site indicates that the oxocarbenium ion of the transition state is stabilized by neighboring-group participation from MgPPi and O5'. A short hydrogen bond forms between Asp 137 and the purine ring analog. Two Mg2+ ions sandwich the pyrophosphate and contact both hydroxyls of the ribosyl analog. The transition-state analog is shielded from bulk solvent by a catalytic loop that moves approximately 25 A to cover the active site and becomes an ordered antiparallel beta-sheet.
Asunto(s)
Difosfatos/química , Inhibidores Enzimáticos/química , Hipoxantina Fosforribosiltransferasa/química , Compuestos de Magnesio/química , Pirimidinonas/química , Pirroles/química , Sitios de Unión , Dominio Catalítico , Cristalización , Cristalografía por Rayos X , Difosfatos/metabolismo , Electrones , Inhibidores Enzimáticos/metabolismo , Humanos , Enlace de Hidrógeno , Hipoxantina Fosforribosiltransferasa/antagonistas & inhibidores , Hipoxantina Fosforribosiltransferasa/metabolismo , Iones , Magnesio/metabolismo , Compuestos de Magnesio/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Nitrógeno/metabolismo , Oxígeno/metabolismo , Fosfatos/metabolismo , Unión Proteica , Estructura Secundaria de Proteína , Pirimidinonas/metabolismo , Pirroles/metabolismo , SolventesRESUMEN
Orotate phosphoribosyltransferase (OMP synthase, EC 2.4.2.10) forms the UMP precursor orotidine 5'-monophophate (OMP) from orotate and alpha-D-5-phosphoribosyl-1-pyrophosphate (PRPP). Here, equilibrium binding, isotope partitioning, and chemical quench studies were used to determine rate and equilibrium constants for the kinetic mechanism. PRPP bound to two sites per dimer with a KD of 33 microM. Binding of OMP and orotate also occurred to a single class of two sites per dimer, with KD values of 3 and 280 microM, respectively. Pyrophosphate binding to two sites was weak with a KD of 960 microM, and in the presence of bound orotate, its affinity for the first site was enhanced 4-fold (KD = 230 microM). Preformed E.OMP, E.PRPP, E.PPi, and E.orotate complexes were trapped as products in isotope partitioning experiments, indicating that each was catalytically competent and confirming a random mechanism. Rapid quench experiments revealed burst kinetics for product formation in both the forward phosphoribosyltransferase and the reverse pyrophosphorolysis reactions. The steady-state rate in the forward reaction was preceded by a burst (nfwd = 1.5/dimer) of at least 300 s-1. In the pyrophosphorolysis reaction, a burst (nrev = 0.7/dimer; k >/= 300 s-1) was also noted. These results allowed us to develop a complete kinetic mechanism for OPRTase, in which a rapid phosphoribosyl transfer reaction at equilibrium is followed by a slow step involving release of product. When the microviscosity, etarel, of the reaction medium was increased with sucrose, the forward kcat decreased in proportion to etarel with a slope of 0.8. In the reverse reaction a more limited dependence of kcat (slope = 0. 3) was observed. On the basis of the known structures of OPRTase, we propose that a highly conserved, catalytically important, solvent-exposed loop descends during catalysis to shield the active site. In the accompanying paper, the slow product release step is shown to relate to movement of the solvent-exposed loop.
Asunto(s)
Orotato Fosforribosiltransferasa/química , Sitios de Unión , Catálisis , Cinética , Ligandos , Fosforribosil Pirofosfato/química , Radioisótopos de Fósforo , Salmonella typhimurium/enzimología , Uridina Monofosfato/análogos & derivados , Uridina Monofosfato/química , ViscosidadRESUMEN
In de novo pyrimidine biosynthesis, orotate phosphoribosyltransferase catalyzes the formation of orotidine 5'-monophosphate (OMP) from orotic acid and alpha-D-5-phosphoribosyl-1-pyrophosphate (PRPP). The known three-dimensional structure of the dimeric enzyme from Salmonella typhimurium is similar to that of other Type I phosphoribosyltransferases (nucleotide synthases) with a solvent-exposed active site atop a Rossman-type nucleotide binding fold. The three-dimensional structure of an enzyme-inhibitor complex [Henriksen et al. (1996) Biochemistry 35, 3803-3809] indicates that one of the two identical solvent-exposed loops can descend to cover the active site of the adjacent subunit of the dimeric enzyme. Catalytically essential residues are known to reside on this loop. In the present work, sensitivity toward limited proteolysis by trypsin confirms that the loop is solvent-exposed. Protection by PRPP and, to a lesser extent, by OMP demonstrates the existence of a second, trypsin-inaccessible, loop position. Two-dimensional 1H-15N NMR relaxation experiments on [alpha-15N]histidine-labeled WT OPRTase yielded backbone 15N T1 and T2 relaxation times and 15N[1H] NOE for His-105 (a loop residue) that are characteristic of small peptides. These results document that the surface loop is highly flexible in the unliganded enzyme. Addition of a hydrolytically stable PRPP analogue to the enzyme resulted in a significant reduction of His-105 peak intensity, indicating a dramatic change in the dynamic properties of the loop backbone in the analogue-ligated enzyme. 1H NMR titrations on histidine C2 protons, coupled with 1H and 31P titrations monitoring the C1H and 5-phosphate PRPP resonances, allowed the quantitation of the rates of loop movement during product release, and relate protein motion to enzymatic catalysis. These results suggest that loop opening and PRPP release is a two-step process, whose overall rate is partially rate-limiting in the reverse pyrophosphorolysis reaction.
Asunto(s)
Modelos Químicos , Orotato Fosforribosiltransferasa/química , Alanina/genética , Catálisis , Difosfatos/química , Endopeptidasas/química , Hidrógeno , Hidrólisis , Lisina/genética , Compuestos de Magnesio/química , Mutagénesis Sitio-Dirigida , Isótopos de Nitrógeno , Resonancia Magnética Nuclear Biomolecular , Orotato Fosforribosiltransferasa/genética , Fosforribosil Pirofosfato/química , Salmonella typhimurium/enzimología , Soluciones , Sulfatos/química , TermodinámicaRESUMEN
BACKGROUND: . Mycobacterium tuberculosis is the single most deadly human pathogen and is responsible for nearly three million deaths every year. Recent elucidation of the mode of action of isoniazid, a frontline antimycobacterial drug, suggests that NAD metabolism is extremely critical for this microorganism. M. tuberculosis depends solely on the de novo pathway to meet its NAD demand. Quinolinic acid phosphoribosyltransferase (QAPRTase), a key enzyme in the de novo biosynthesis of NAD, provides an attractive target for designing novel antitubercular drugs. RESULTS: . The X-ray crystal structure of the M. tuberculosis QAPRTase apoenzyme has been determined by multiple isomorphous replacement at 2.4 A resolution. Structures of the enzyme have also been solved in complex with the substrate quinolinic acid (QA), the inhibitory QA analog phthalic acid (PA), the product nicotinate mononucleotide (NAMN), and as a ternary complex with PA and a substrate analog, 5-phosphoribosyl-1-(beta-methylene)pyrophosphate (PRPCP). The structure of the nonproductive QAPRTase-PA-PRPCP Michaelis complex reveals a 5-phosphoribosyl-1-pyrophosphate-binding site that is different from the one observed in type I phosphoribosyltransferases (PRTases). The type II PRTase active site of QAPRTase undergoes conformational changes that appear to be important in determining substrate specificity and eliciting productive catalysis. CONCLUSIONS: . QAPRTase is the only known representative of the type II PRTase fold, an unusual alpha/beta barrel, and appears to represent convergent evolution for PRTase catalysis. The active site of type II PRTase bears little resemblance to the better known type I enzymes.
Asunto(s)
Antituberculosos/síntesis química , Mycobacterium tuberculosis/enzimología , Pentosiltransferasa/química , Secuencia de Aminoácidos , Antituberculosos/farmacología , Cristalografía por Rayos X , Diseño de Fármacos , Humanos , Datos de Secuencia Molecular , Mycobacterium tuberculosis/efectos de los fármacos , Mononucleótido de Nicotinamida/análogos & derivados , Mononucleótido de Nicotinamida/metabolismo , Pentosiltransferasa/metabolismo , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) catalyzes the reversible formation of IMP and GMP from their respective bases hypoxanthine (Hx) and guanine (Gua) and the phosphoribosyl donor 5-phosphoribosyl-1-pyrophosphate (PRPP). The net formation and cleavage of the nucleosidic bond requires removal/addition of a proton at the purine moiety, allowing enzymic catalysis to reduce the energy barrier associated with the reaction. The pH profile of kcat for IMP pyrophosphorolysis revealed an essential acidic group with pKa of 7.9 whereas those for IMP or GMP formation indicated involvement of essential basic groups. Based on the crystal structure of human HGPRTase, protonation/deprotonation is likely to occur at N7 of the purine ring, and Lys 165 or Asp 137 are each candidates for the general base/acid. We have constructed, purified, and kinetically characterized two mutant HGPRTases to test this hypothesis. D137N displayed an 18-fold decrease in kcat for nucleotide formation with Hx as substrate, a 275-fold decrease in kcat with Gua, and a 500-fold decrease in kcat for IMP pyrophosphorolysis. D137N also showed lower KD values for nucleotides and PRPP. The pH profiles of kcat for D137N were severely altered. In contrast to D137N, the kcat for K165Q was decreased only 2-fold in the forward reaction and was slightly increased in the reverse reaction. The Km and KD values showed that K165Q interacts with substrates more weakly than does the wild-type enzyme. Pre-steady-state experiments with K165Q indicated that the phosphoribosyl transfer step was fast in the forward reaction, as observed with the wild type. In contrast, D137N showed slower phosphoribosyl transfer chemistry, although guanine (3000-fold reduction) was affected much more than hypoxanthine (32-fold reduction). In conclusion, Asp137 acts as a general catalytic acid/base for HGPRTase and Lys165 makes ground-state interactions with substrates.
Asunto(s)
Ácido Aspártico/metabolismo , Hipoxantina Fosforribosiltransferasa/metabolismo , Sustitución de Aminoácidos/genética , Asparagina/genética , Asparagina/metabolismo , Ácido Aspártico/química , Ácido Aspártico/genética , Catálisis , Glutamina/genética , Glutamina/metabolismo , Guanosina Monofosfato/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Hipoxantina Fosforribosiltransferasa/química , Hipoxantina Fosforribosiltransferasa/genética , Inosina Monofosfato/metabolismo , Cinética , Ligandos , Lisina/genética , Lisina/metabolismo , Mutagénesis Sitio-Dirigida , Reacción en Cadena de la PolimerasaRESUMEN
Nicotinic acid phosphoribosyltransferase (NAPRTase; EC 2.4.2.11) is a facultative ATPase that uses the energy of ATP hydrolysis to drive the synthesis of nicotinate mononucleotide and pyrophosphate from nicotinic acid (NA) and phosphoribosyl pyrophosphate (PRPP). To learn how NAPRTase uses this hydrolytic energy, we have further delineated the kinetic mechanism using steady-state and pre-steady-state kinetics, equilibrium binding, and isotope trapping. NAPRTase undergoes covalent phosphorylation by bound ATP at a rate of 30 s-1. The phosphoenzyme (E-P) binds PRPP with a KD of 0.6 microM, a value 2000-fold lower than that measured for the nonphosphorylated enzyme. The minimal rate constant for PRPP binding to E-P is 0.72 x 10(5) M-1 s-1. Isotope trapping shows that greater than 90% of bound PRPP partitions toward product upon addition of NA. Binding of NA to E-P.PRPP is rapid, kon >/= 7.0 x 10(6) M-1 s-1, and is followed by rapid formation of NAMN and PPi, k >/= 500 s-1. After product formation, E-P undergoes hydrolytic cleavage, k = 6.3 s-1, and products NAMN, PPi, and Pi are released. Quenching from the steady state under Vmax conditions indicates that slightly less than half the enzyme is in phosphorylated forms. To account for this finding, we propose that one step in the release of products is as slow as 5.2 s-1 and, together with the E-P cleavage step, codetermines the overall kcat of 2.3 s-1 at 22 degrees C. Energy coupling by NAPRTase involves two strategies frequently proposed for ATPases of macromolecular recognition and processing. First, E-P has a 10(3)-fold higher affinity for substrates than does nonphosphorylated enzyme, allowing the E-P to bind substrate from low concentration and nonphosphorylated enzyme to expel products against a high concentration. Second, the kinetic pathway follows "rules" [Jencks, W. P. (1989) J. Biol. Chem. 264, 18855-18858] that minimize unproductive alternative reaction pathways. However, an analysis of reaction schemes based on these strategies suggests that such nonvectorial reactions are intrinsically inefficient in ATP use.
Asunto(s)
Transferencia de Energía , Pentosiltransferasa/química , Pentosiltransferasa/metabolismo , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Sitios de Unión , Difosfatos/metabolismo , Hidrólisis , Marcaje Isotópico/métodos , Cinética , Niacina/metabolismo , Mononucleótido de Nicotinamida/análogos & derivados , Mononucleótido de Nicotinamida/biosíntesis , Mononucleótido de Nicotinamida/metabolismo , Fosforilación , Especificidad por SustratoRESUMEN
Nicotinic acid phosphoribosyltransferase (NAPRTase; EC 2.4.2.11) forms nicotinic acid mononucleotide (NAMN) and PPi from 5-phosphoribosyl 1-pyrophosphate (PRPP) and nicotinic acid (NA). The Vmax NAMN synthesis activity of the Salmonella typhimurium enzyme is stimulated about 10-fold by ATP, which, when present, is hydrolyzed to ADP and Pi in 1:1 stoichiometry with NAMN formed. The overall NAPRTase reaction involves phosphorylation of a low-affinity form of the enzyme by ATP, followed by generation of a high-affinity form of the enzyme, which then binds substrates and produces NAMN. Hydrolysis of E-P then regenerates the low-affinity form of the enzyme with subsequent release of products. Our earlier studies [Gross, J., Rajavel, M., Segura, E., and Grubmeyer, C. (1996) Biochemistry 35, 3917-3924] have shown that His-219 becomes phosphorylated in the N1 (pi) position by ATP. Here, we have mutated His-219 to glutamate and asparagine and determined the properties of the purified mutant enzymes. The mutant NAPRTases fail to carry out ATPase, autophosphorylation, or ADP/ATP exchanges seen with wild-type (WT) enzyme. The mutants do catalyze the slow formation of NAMN in the absence of ATP with rates and KM values similar to those of WT. In striking contrast to WT, NAMN formation by the mutant enzymes is competitively inhibited by ATP. Thus, the NAMN synthesis reaction may occur at a site overlapping that for ATP. Previous studies suggest that the yeast NAPRTase does not catalyze NAMN synthesis in the absence of ATP. We have cloned, overexpressed, and purified the yeast enzyme and report its kinetic properties, which are similar to those of the bacterial enzyme.
Asunto(s)
Mutagénesis Insercional , Pentosiltransferasa/antagonistas & inhibidores , Pentosiltransferasa/genética , Adenosina Trifosfato/metabolismo , Adenosina Trifosfato/farmacología , Secuencia de Aminoácidos , Sitios de Unión/genética , Fenómenos Químicos , Química Física , Difosfatos/metabolismo , Hidrólisis , Datos de Secuencia Molecular , Mononucleótido de Nicotinamida/análogos & derivados , Mononucleótido de Nicotinamida/antagonistas & inhibidores , Mononucleótido de Nicotinamida/biosíntesis , Pentosiltransferasa/biosíntesis , Pentosiltransferasa/aislamiento & purificación , Pentosiltransferasa/metabolismo , Fosforribosil Pirofosfato/metabolismo , Fosforilación , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Salmonella typhimurium/enzimología , Salmonella typhimurium/genética , Especificidad por Sustrato/genética , TripsinaRESUMEN
Hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) is the locus of Lesch-Nyhan syndrome, the activator of the prodrugs 6-mercaptopurine and allopurinol, and a target for antiparasitic chemotherapy. The three-dimensional structure of the recombinant human enzyme in complex with GMP has recently been solved [Eads, J., Scapin, G., Xu, Y., Grubmeyer, C., & Sacchettini, J. C. (1994) Cell 78, 325-334]. Here, ligand binding, pre-steady state kinetics, isotope trapping, and isotope exchange experiments are presented which detail the sequential kinetic mechanism of the enzyme. In the forward reaction, in which a base (hypoxanthine or guanine) reacts with PRPP to form nucleoside monophosphate and PPi, binding of PRPP precedes that of the base, and in the reverse direction, IMP binds first. Compared to k(cat), phosphoribosyl group transfer is rapid in both the forward (131 vs 6.0 s(-1)) and reverse (9 vs 0.17 s(-1)) directions. In the forward direction, product pyrophosphate dissociates rapidly (> 12 s(-1)) followed by release of IMP (6.0 s(-1)). In the reverse direction, Hx dissociates rapidly (9.5 s(-1)) and PRPP dissociates slowly (0.24 s(-1)). The more rapid rate of utilization of guanine than hypoxanthine in the forward reaction is the result of the faster release of product GMP rather than the result of differences in the rate of the chemical step. The kinetic mechanism, with rapid chemistry and slow product dissociation, accounts for the previously observed ability of the alternative product guanine to stimulate, rather than inhibit, the pyrophosphorolysis of IMP. The overall equilibrium for the hypoxanthine phosphoribosyl transfer reaction lies far toward nucleotide product (Keq approximately 1.6 x 10(5)), at the high end for PRPP-linked nucleotide formation. The three-dimensional structure of the HGPRTase x IMP complex has been solved to 2.4 A resolution and is isomorphous with the GMP complex. The results of the ligand binding and kinetic studies are discussed in light of the structural data.
Asunto(s)
Hipoxantina Fosforribosiltransferasa/metabolismo , Fosforribosil Pirofosfato/metabolismo , Guanina/metabolismo , Guanosina Monofosfato/metabolismo , Humanos , Hipoxantina/metabolismo , Inosina Monofosfato/metabolismo , Cinética , Modelos Químicos , Modelos Moleculares , Conformación Proteica , Proteínas Recombinantes/metabolismoRESUMEN
BACKGROUND: Quinolinic acid (QA) is a neurotoxin and has been shown to be present at high levels in the central nervous system of patients with certain diseases, such as AIDS and meningitis. The enzyme quinolinic acid phosphoribosyltransferase (QAPRTase) provides the only route for QA metabolism and is also an essential step in de novo NAD biosynthesis. QAPRTase catalyzes the synthesis of nicotinic acid mononucleotide (NAMN) from QA and 5-phosphoribosyl-1-pyrophosphate (PRPP). The structures of several phosphoribosyltransferases (PRTases) have been reported, and all have shown a similar fold of a five-strandard beta sheet surrounded by four alpha helices. A conserved sequence motif of 13 residues is common to these 'type I' PRTases but is not observed in the QAPRTase sequence, suggestive of a different fold for this enzyme. RESULTS: The crystal structure of QAPRTase from Salmonella typhimurium has been determined with bound QA to 2.8 A resolution, and with bound NAMN to 3.0 A resolution. Most significantly, the enzyme shows a completely novel fold for a PRTase enzyme comprising a two-domain structure: a mixed alpha/beta N-terminal domain and an alpha/beta barrel-like domain containing seven beta strands. The active site is located at the C-terminal ends of the beta strands of the alpha/beta barrel, and is bordered by the N-terminal domain of the second subunit of the dimer. The active site is largely composed of a number of conserved charged residues that appear to be important for substrate binding and catalysis. CONCLUSIONS: The seven-stranded alpha/beta-barrel domain of QAPRTase is very similar in structure to the eight-stranded alpha/beta-barrel enzymes. The structure shows a phosphate-binding site that appears to be conserved among many alpha/beta-barrel enzymes including indole-3-glycerol phosphate synthase and flavocytochrome b2. The new fold observed here demonstrates that the PRTase enzymes have evolved their similar chemistry from at least two completely different protein architectures.
Asunto(s)
Pentosiltransferasa/química , Salmonella typhimurium/enzimología , Secuencia de Aminoácidos , Sitios de Unión , Clonación Molecular , Cristalografía por Rayos X , Dimerización , Modelos Químicos , Modelos Moleculares , Datos de Secuencia Molecular , Mononucleótido de Nicotinamida/análogos & derivados , Mononucleótido de Nicotinamida/química , Mononucleótido de Nicotinamida/metabolismo , Fosfatos/metabolismo , Conformación Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Ácido Quinolínico/química , Ácido Quinolínico/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Homología de Secuencia de AminoácidoRESUMEN
PITALRE is a human protein kinase belonging to the cell division cycle 2 (CDC2) kinase family, and is the catalytic subunit of a multimeric complex that contains several cellular proteins. PITALRE complexes from several cell lines and tissues phosphorylate retinoblastoma protein and myelin basic protein (MBP). In the present work, we have found that MBP is phosphorylated by PITALRE complexes on both Ser and Thr residues. Two different antibodies raised to PITALRE purified virtually identical kinase activities, as analysed by MBP phosphopeptide mapping and phosphoamino acid analysis. We have identified the proline-directed residue Ser-162 of MBP as a major phosphorylation site for PITALRE. In addition, our results suggest that one of the two MBP proline-directed threonine residues, Thr-97, is also selectively phosphorylated by PITALRE. These data, together with analysis of different peptide substrates derived from sites on MBP that are phosphorylated by PITALRE, indicate that PITALRE is a Ser/Thr proline-directed kinase. In addition, our results show that PITALRE has a substrate site specificity distinguishable from those of the CDC2 and cyclin-dependent kinase 2 (CDK2).
Asunto(s)
Proteína Básica de Mielina/metabolismo , Proteínas Quinasas/metabolismo , Secuencia de Aminoácidos , Cromatografía Líquida de Alta Presión , Quinasa 9 Dependiente de la Ciclina , Electroforesis en Gel Bidimensional , Células HeLa , Humanos , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Mapeo Peptídico , Fosfopéptidos/química , Fosforilación , Análisis de Secuencia , Tripsina/metabolismoRESUMEN
Nicotinic acid phosphoribosyltransferase (NAPRTase;EC 2.4.2.11) couples stoichiometric ATP hydrolysis with formation of nicotinate mononucleotide (NAMN) from nicotinic acid and alpha-D-5-phosphoribosyl 1-pyrophosphate (PRPP). Trypsin rapidly inactivated the ATPase and NAMN synthesis activities of NAPRTase in parallel, with cleavages at Arg-384 and Lys-374 of the 399-residue protein. ATP and PRPP each provided protection against tryptic cleavage. Limited chymotryptic proteolysis of NAPRTase exhibited very similar behavior, with specific cleavage at Phe-382 and protection by substrates. Results suggest that a solvent-exposed loop encompassing Lys-374, Phe-382, and Arg-384 is protected by ATP- or PRPP-induced conformational changes. The ability of ATP to protect even under conditions in which enzyme phosphorylation was prevented by EDTA provides evidence for a distinct ATP-induced protein conformation that acts as an intermediate in energy coupling.
Asunto(s)
Adenosina Trifosfato/metabolismo , Pentosiltransferasa/química , Pentosiltransferasa/metabolismo , Conformación Proteica , Salmonella typhimurium/enzimología , Adenosina Trifosfato/farmacología , Secuencia de Aminoácidos , Cromatografía por Intercambio Iónico , Clonación Molecular , Expresión Génica , Cinética , Espectrometría de Masas , Datos de Secuencia Molecular , Pentosiltransferasa/aislamiento & purificación , Fragmentos de Péptidos/química , Fragmentos de Péptidos/aislamiento & purificación , Mapeo Peptídico , Fosforilación , Conformación Proteica/efectos de los fármacos , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/enzimología , Homología de Secuencia de Aminoácido , TripsinaRESUMEN
Energy coupling between ATP hydrolysis and other enzyme reactions requires the phosphorylation of substrate-derived intermediates, or the existence of enzyme-derived intermediates capable of storage and transfer of energy. Salmonella typhimurium nicotinic acid phosphoribosyltransferase (NAPRTase, EC 2.4.2.11) couples net ATP hydrolysis to formation of NAMN and PPi from alpha-PRPP and nicotinic acid [Vinitsky, A., & Grubmeyer, C (1993) J. Biol. Chem. 268, 26004-26010]. In the current work, we have determined that the enzyme reacts with ATP to produce a covalently phosphorylated form of the enzyme (E-P), which is common to both the ATPase and NAMN synthesis functions of NAPRTase. We have isolated E-P and verified its catalytic competence. E-P showed acid lability and base stability, diagnostic of a phosphoramidate linkage. Pyridine and hydroxylamine-catalyzed hydrolysis of E-P gave second-order rate constants consistent with published values for phosphohistidine. Two-dimensional thin-layer chromatography of alkaline-hydrolyzed E-32P showed that the phosphorylated residue co-migrated with authentic 1-phosphohistidine. Chymotrypsin and trypsin proteolysis followed by HPLC and peptide sequencing localized the phosphopeptide to Ala-210 to Phe-222 of the 399-residue protein. This peptide contains a single histidine residue, His-219. NAPRTase phosphorylated at His-219 is an intermediate in the energy transduction mechanism of NAPRTase.