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1.
Arch Biochem Biophys ; 413(1): 23-31, 2003 May 01.
Article in English | MEDLINE | ID: mdl-12706338

ABSTRACT

Escherichia coli asparagine synthetase B (AS-B) catalyzes the formation of asparagine from aspartate in an ATP-dependent reaction for which glutamine is the in vivo nitrogen source. In an effort to reconcile several different kinetic models that have been proposed for glutamine-dependent asparagine synthetases, we have used numerical methods to investigate the kinetic mechanism of AS-B. Our simulations demonstrate that literature proposals cannot reproduce the glutamine dependence of the glutamate/asparagine stoichiometry observed for AS-B, and we have therefore developed a new kinetic model that describes the behavior of AS-B more completely. The key difference between this new model and the literature proposals is the inclusion of an E.ATP.Asp.Gln quaternary complex that can either proceed to form asparagine or release ammonia through nonproductive glutamine hydrolysis. The implication of this model is that the two active sites in AS-B become coordinated only after formation of a beta-aspartyl-AMP intermediate in the synthetase site of the enzyme. The coupling of glutaminase and synthetase activities in AS is therefore different from that observed in all other well-characterized glutamine-dependent amidotransferases.


Subject(s)
Carbon-Nitrogen Ligases with Glutamine as Amide-N-Donor/metabolism , Escherichia coli/enzymology , Ammonia/metabolism , Asparagine/biosynthesis , Aspartate-Ammonia Ligase/chemistry , Aspartate-Ammonia Ligase/metabolism , Carbon-Nitrogen Ligases with Glutamine as Amide-N-Donor/chemistry , Catalytic Domain , Glutamine/metabolism , Kinetics , Models, Biological , Models, Molecular , Protein Conformation
2.
J Med Chem ; 44(24): 4114-24, 2001 Nov 22.
Article in English | MEDLINE | ID: mdl-11708914

ABSTRACT

The melanocortin receptors are G-protein coupled receptors (GPCRs) that activate the cAMP signal transduction pathway and are stimulated by the melanocortin agonist alpha-melanocyte stimulating hormone (alpha-MSH). Members of these melanocortin receptors are antagonized by agouti (ASP) and agouti-related protein (AGRP), which are the only known endogenous antagonists of GPCRs identified to date. Structure-function studies of the hAGRP(109-118) decapeptide, Tyr-c[Cys-Arg-Phe-Phe-Asn-Ala-Phe-Cys]-Tyr-NH(2), by replacing the 26-membered disulfide Cys(2)-Cys(9) ring with lactam bridges resulted in the identification of a novel peripheral skin melanocortin-1 receptor (MC1R) antagonist. This antagonist, Tyr-c[Glu-Arg-Phe-Phe-Asn-Ala-Phe-Dpr]-Tyr-NH(2), possesses a 27-membered ring with the lactam bridge being formed from the Calpha-carboxyl moiety of Glu (instead of the typical side chain carboxyl moiety) with the amine of the diaminopropionic acid (Dpr) residue. This mouse MC1 receptor antagonist (pA(2) = 5.9) is also an antagonist at the brain melanocortin-4 receptor (pA(2) = 6.9), with no observable pharmacology at the melanocortin-3 or -5 receptors. This MC1R hAGRP(109-118) based decapeptide is novel in that AGRP(83-132) itself does not bind to, agonize, or antagonize the skin MC1R. Structural analysis has been performed using two-dimensional (1)H NMR and computer-assisted molecular modeling (CAMM) techniques in attempts to identify structural features of this Tyr-c[Glu-Arg-Phe-Phe-Asn-Ala-Phe-Dpr]-Tyr-NH(2) (cyclo Glu alphaCOOH-Dpr betaNH) peptide that can differentially result in antagonist versus agonist properties at the mMC1R.


Subject(s)
Intercellular Signaling Peptides and Proteins , Lactams/chemical synthesis , Peptides, Cyclic/chemical synthesis , Proteins/chemistry , Receptor, Melanocortin, Type 3 , Receptors, Corticotropin/antagonists & inhibitors , Agouti Signaling Protein , Agouti-Related Protein , Amino Acid Sequence , Animals , Cell Line , Humans , Lactams/chemistry , Lactams/pharmacology , Magnetic Resonance Spectroscopy , Mice , Models, Molecular , Molecular Conformation , Molecular Sequence Data , Peptide Fragments/chemistry , Peptides, Cyclic/chemistry , Peptides, Cyclic/pharmacology , Receptors, Corticotropin/agonists , Receptors, Melanocortin , Skin/chemistry , Structure-Activity Relationship , Transfection
3.
Biochemistry ; 40(37): 11168-75, 2001 Sep 18.
Article in English | MEDLINE | ID: mdl-11551215

ABSTRACT

Asparagine synthetase catalyzes the ATP-dependent formation of L-asparagine from L-aspartate and L-glutamine, via a beta-aspartyl-AMP intermediate. Since interfering with this enzyme activity might be useful for treating leukemia and solid tumors, we have sought small-molecule inhibitors of Escherichia coli asparagine synthetase B (AS-B) as a model system for the human enzyme. Prior work showed that L-cysteine sulfinic acid competitively inhibits this enzyme by interfering with L-aspartate binding. Here, we demonstrate that cysteine sulfinic acid is also a partial substrate for E. coli asparagine synthetase, acting as a nucleophile to form the sulfur analogue of beta-aspartyl-AMP, which is subsequently hydrolyzed back to cysteine sulfinic acid and AMP in a futile cycle. While cysteine sulfinic acid did not itself constitute a clinically useful inhibitor of asparagine synthetase B, these results suggested that replacing this linkage by a more stable analogue might lead to a more potent inhibitor. A sulfoximine reported recently by Koizumi et al. as a competitive inhibitor of the ammonia-dependent E. coli asparagine synthetase A (AS-A) [Koizumi, M., Hiratake, J., Nakatsu, T., Kato, H., and Oda, J. (1999) J. Am. Chem. Soc. 121, 5799-5800] can be regarded as such a species. We found that this sulfoximine also inhibited AS-B, effectively irreversibly. Unlike either the cysteine sulfinic acid interaction with AS-B or the sulfoximine interaction with AS-A, only AS-B productively engaged in asparagine synthesis could be inactivated by the sulfoximine; free enzyme was unaffected even after extended incubation with the sulfoximine. Taken together, these results support the notion that sulfur-containing analogues of aspartate can serve as platforms for developing useful inhibitors of AS-B.


Subject(s)
Adenosine Monophosphate/pharmacology , Asparagine/biosynthesis , Carbon-Nitrogen Ligases with Glutamine as Amide-N-Donor/antagonists & inhibitors , Escherichia coli/enzymology , Methionine Sulfoximine/pharmacology , Adenosine Monophosphate/analogs & derivatives , Adenosine Triphosphate/metabolism , Cysteine/analogs & derivatives , Cysteine/pharmacology , Enzyme Inhibitors/pharmacology , Hydrolysis , Methionine Sulfoximine/analogs & derivatives , Models, Chemical , Neurotransmitter Agents , Nuclear Magnetic Resonance, Biomolecular , Phosphorus Isotopes , Spectrometry, Mass, Electrospray Ionization
4.
J Org Chem ; 66(19): 6381-8, 2001 Sep 21.
Article in English | MEDLINE | ID: mdl-11559190

ABSTRACT

Fluorine-containing derivatives of amino acids are assuming increasing importance as probes of biological function and enzyme mechanism. We now report a new, flexible route to enantiomerically pure L-4,4-difluoroglutamic acid that exploits the addition of difluorinated nucleophiles to configurationally stable alpha-aminoaldehydes. Conversion of the difluorinated adducts to L-4,4-difluoroglutamic acid can be accomplished in three steps by Barton-McCombie dehydroxylation and acid hydrolysis.


Subject(s)
Aldehydes/chemistry , Glutamates/chemical synthesis , Aspartate-Ammonia Ligase/chemistry , Aspartate-Ammonia Ligase/metabolism , Glutamates/chemistry , Glutamates/metabolism , Hydrolysis , Hydroxylation , Molecular Conformation
5.
Inorg Chem ; 40(8): 1837-45, 2001 Apr 09.
Article in English | MEDLINE | ID: mdl-11312740

ABSTRACT

Important structural and mechanistic details concerning the non-heme, low-spin Fe(III) center in nitrile hydratase (NHase) remain poorly understood. We now report projection unrestricted Hartree-Fock (PUHF) calculations on the spin preferences of a series of inorganic complexes in which Fe(III) is coordinated by a mixed set of N/S ligands. Given that many of these compounds have been prepared as models of the NHase metal center, this study has allowed us to evaluate this computational approach as a tool for future calculations on the electronic structure of the NHase Fe(III) center itself. When used in combination with the INDO/S semiempirical model, the PUHF method correctly predicts the experimentally observed spin state for 12 of the 13 Fe(III)-containing complexes studied here. The one compound for which there is disagreement between our theoretical calculations and experimental observation exhibits temperature-dependent spin behavior. In this case, the failure of the PUHF-INDO/S approach may be associated with differences between the structure of the Fe(III) complex present under the conditions used to measure the spin preference and that observed by X-ray crystallography. A preliminary analysis of the role of the N/S ligands and coordination geometry in defining the Fe(III) spin preferences in these complexes has also been undertaken by computing the electronic properties of the lowest energy Fe(III) spin states. While any detailed interpretation of our results is constrained both by the limited set of well-characterized Fe(III) complexes used in this study and by the complicated dependence of Fe(III) spin preference upon metal-ligand interactions and coordination geometry, these PUHF-INDO/S calculations support the hypothesis that the deprotonated amide nitrogens coordinating the metal stabilize the low-spin Fe(III) ground state seen in NHase. Strong evidence that the sulfur ligands exclusively define the Fe(III) spin state preference by forming metal-ligand bonds with significant covalent character is not provided by these computational studies. This might, however, reflect limitations in modeling these systems at the INDO/S level of theory.


Subject(s)
Ferric Compounds/chemistry , Hydro-Lyases/chemistry , Models, Chemical , Computer Simulation , Ferric Compounds/metabolism , Hydro-Lyases/metabolism , Protein Conformation
6.
Biochemistry ; 38(49): 16146-57, 1999 Dec 07.
Article in English | MEDLINE | ID: mdl-10587437

ABSTRACT

Asparagine synthetase B catalyzes the assembly of asparagine from aspartate, Mg(2+)ATP, and glutamine. Here, we describe the three-dimensional structure of the enzyme from Escherichia colidetermined and refined to 2.0 A resolution. Protein employed for this study was that of a site-directed mutant protein, Cys1Ala. Large crystals were grown in the presence of both glutamine and AMP. Each subunit of the dimeric protein folds into two distinct domains. The N-terminal region contains two layers of antiparallel beta-sheet with each layer containing six strands. Wedged between these layers of sheet is the active site responsible for the hydrolysis of glutamine. Key side chains employed for positioning the glutamine substrate within the binding pocket include Arg 49, Asn 74, Glu 76, and Asp 98. The C-terminal domain, responsible for the binding of both Mg(2+)ATP and aspartate, is dominated by a five-stranded parallel beta-sheet flanked on either side by alpha-helices. The AMP moiety is anchored to the protein via hydrogen bonds with O(gamma) of Ser 346 and the backbone carbonyl and amide groups of Val 272, Leu 232, and Gly 347. As observed for other amidotransferases, the two active sites are connected by a tunnel lined primarily with backbone atoms and hydrophobic and nonpolar amino acid residues. Strikingly, the three-dimensional architecture of the N-terminal domain of asparagine synthetase B is similar to that observed for glutamine phosphoribosylpyrophosphate amidotransferase while the molecular motif of the C-domain is reminiscent to that observed for GMP synthetase.


Subject(s)
Aspartate-Ammonia Ligase/chemistry , Escherichia coli/enzymology , Adenosine Monophosphate/chemistry , Adenosine Monophosphate/metabolism , Amidophosphoribosyltransferase/chemistry , Aspartate-Ammonia Ligase/metabolism , Binding Sites , Crystallography, X-Ray , Models, Molecular , Molecular Sequence Data , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Protein Conformation , Protein Structure, Secondary , Protein Structure, Tertiary , Structure-Activity Relationship , Substrate Specificity
7.
Naunyn Schmiedebergs Arch Pharmacol ; 359(3): 168-77, 1999 Mar.
Article in English | MEDLINE | ID: mdl-10208303

ABSTRACT

Several carbostyril-based beta-agonists have been shown to bind tightly to and slowly dissociate from the beta2-adrenoceptor (beta2AR). In the present study, the structural features of 8-hydroxy-5-[2-[(1-phenyl-2-methylprop-2-yl)amino]-1-hydroxyethyl] -carbostyril (11a) which contribute to its binding properties at the beta2AR were investigated using a series of synthesized analogs. The k(off), estimated by the rate of cAMP decline in DDT1 MF-2 (DDT) cells with a reduced receptor density, Ki and ligand-induced receptor reductions were determined. All of the derivatives stimulated cAMP accumulation in DDT cells in the sub to mid nanomolar range and elicited the same maximal stimulation as (-)isoproterenol. Derivatives of 11a with side chain N-substitutions comprising 2-methylbutyl, phenylethyl and isopropyl had higher k(off)-values and lower affinities as compared to 11a. Increasing the number of methylenes between the side chain tertiary alpha carbon and phenyl from 1 in 11a to 3 or reducing the number to 0 also resulted in derivatives with higher k(off)- and Ki-values. In addition, replacement of the 8-hydroxycarbostyril nucleus of 11a with catechol reduced the affinity of the compound for the beta2AR by 48-fold and increased its k(off). Only those derivatives with the lowest k(off)-values induced a decrease in the receptor density of DDT cell membranes following a preincubation and extensive washing. The data show that the 8-hydroxycarbostyril nucleus in conjunction with substitutions on the tertiary alpha carbon of the side chain and positioning of the phenyl group are important characteristics determining the high affinity and slow dissociation of 11a from the beta2AR.


Subject(s)
Adrenergic beta-Agonists/metabolism , Catechols/chemistry , Cyclic AMP/analysis , Hydroxyquinolines/metabolism , Phosphoric Diester Hydrolases/metabolism , Quinolones , Receptors, Adrenergic, beta-2/metabolism , Adrenergic beta-Agonists/chemical synthesis , Cell Culture Techniques , Cell Line , Hydroxyquinolines/chemistry , Isoproterenol/pharmacology , Ligands , Linear Models , Structure-Activity Relationship , Time Factors
8.
Biochemistry ; 38(12): 3677-82, 1999 Mar 23.
Article in English | MEDLINE | ID: mdl-10090755

ABSTRACT

Incubation of Escherichia coli asparagine synthetase B (AS-B) with [14C]-L-glutamine gives a covalent adduct that can be isolated. Radiolabeled protein is not observed (i) when the wild-type enzyme is incubated with 6-diazo-5-oxo-L-norleucine (DON) prior to reaction with [14C]glutamine or (ii) when the C1A AS-B mutant is incubated with [14C]-L-glutamine. Both of these alterations eliminate the ability of the enzyme to utilize glutamine but do not affect ammonia-dependent asparagine synthesis. Formation of the covalent adduct therefore depends on the presence of the N-terminal active site cysteine, which has been shown to be essential for glutamine-dependent activity in this and other class II amidotransferases. The amount of covalent adduct exhibits saturation behavior with increasing concentrations of L-glutamine. The maximum observed quantity of this intermediate is consistent with its involvement on the main pathway of glutamine hydrolysis. The chemical properties of the isolable covalent adduct are consistent with those anticipated for the gamma-glutamyl thioester that has been proposed as an intermediate in the AS-B-catalyzed conversion of glutamine to glutamate. The covalent adduct is acid-stable but is labile under alkaline conditions. On the basis of the measured rates of formation and breakdown of this intermediate, it is kinetically competent to participate in the normal catalytic mechanism. These studies represent the first description of a thioester intermediate for any class II amidotransferase and represent an important step in gaining further insight into the kinetic and chemical mechanisms of AS-B.


Subject(s)
Aspartate-Ammonia Ligase/chemistry , Glutaminase/chemistry , Glutamine/chemistry , Aspartate-Ammonia Ligase/metabolism , Carbon Radioisotopes , Escherichia coli/enzymology , Glutaminase/metabolism , Glutamine/metabolism , Hydroxylamine/metabolism , Kinetics
9.
Biochemistry ; 37(38): 13230-8, 1998 Sep 22.
Article in English | MEDLINE | ID: mdl-9748330

ABSTRACT

Escherichia coli asparagine synthetase B (AS-B) catalyzes the synthesis of asparagine from aspartate, glutamine, and ATP. A combination of kinetic, isotopic-labeling, and stoichiometry studies have been performed to define the nature of nitrogen transfer mediated by AS-B. The results of initial rate studies were consistent with initial binding and hydrolysis of glutamine to glutamate plus enzyme-bound ammonia. The initial velocity results were equally consistent with initial binding of ATP and aspartate prior to glutamine binding. However, product inhibition studies were only consistent with the latter pathway. Moreover, isotope-trapping studies confirmed that the enzyme-ATP-aspartate complex was kinetically competent. Studies using 18O-labeled aspartate were consistent with formation of a beta-aspartyl-AMP intermediate, and stoichiometry studies revealed that 1 equiv of this intermediate formed on the enzyme in the absence of a nitrogen source. Taken together, our results are most consistent with initial formation of beta -aspartyl-AMP intermediate prior to glutamine binding. This sequence leaves open many possibilities for the chemical mechanism of nitrogen transfer.


Subject(s)
Aspartate-Ammonia Ligase/metabolism , Escherichia coli/enzymology , Adenosine Monophosphate/chemistry , Adenosine Monophosphate/metabolism , Asparagine/chemistry , Asparagine/metabolism , Aspartate-Ammonia Ligase/antagonists & inhibitors , Aspartate-Ammonia Ligase/chemistry , Aspartic Acid/chemistry , Aspartic Acid/metabolism , Bacterial Proteins/antagonists & inhibitors , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Binding, Competitive , Glutamine/chemistry , Glutamine/metabolism , Isotope Labeling , Kinetics , Oxygen Isotopes , Substrate Specificity
11.
Article in English | MEDLINE | ID: mdl-9559053

ABSTRACT

The enzymatic synthesis of asparagine is an ATP-dependent process that utilizes the nitrogen atom derived from either glutamine or ammonia. Despite a long history of kinetic and mechanistic investigation, there is no universally accepted catalytic mechanism for this seemingly straightforward carboxyl group activating enzyme, especially as regards those steps immediately preceding amide bond formation. This chapter considers four issues dealing with the mechanism: (a) the structural organization of the active site(s) partaking in glutamine utilization and aspartate activation; (b) the relationship of asparagine synthetase to other amidotransferases; (c) the way in which ATP is used to activate the beta-carboxyl group; and (d) the detailed mechanism by which nitrogen is transferred.


Subject(s)
Aspartate-Ammonia Ligase/metabolism , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Ammonia/metabolism , Aspartate-Ammonia Ligase/antagonists & inhibitors , Aspartate-Ammonia Ligase/classification , Aspartic Acid/metabolism , Binding Sites , Eukaryotic Cells/enzymology , Glutamine/metabolism , Molecular Sequence Data , Prokaryotic Cells/enzymology , Sequence Homology, Amino Acid
12.
J Biol Chem ; 272(19): 12384-92, 1997 May 09.
Article in English | MEDLINE | ID: mdl-9139684

ABSTRACT

Site-directed mutagenesis and kinetic studies have been employed to identify amino acid residues involved in aspartate binding and transition state stabilization during the formation of beta-aspartyl-AMP in the reaction mechanism of Escherichia coli asparagine synthetase B (AS-B). Three conserved amino acids in the segment defined by residues 317-330 appear particularly crucial for enzymatic activity. For example, when Arg-325 is replaced by alanine or lysine, the resulting mutant enzymes possess no detectable asparagine synthetase activity. The catalytic activity of the R325A AS-B mutant can, however, be restored to about 1/6 of that of wild-type AS-B by the addition of guanidinium HCl (GdmHCl). Detailed kinetic analysis of the rescued activity suggests that Arg-325 is involved in stabilization of a pentacovalent intermediate leading to the formation beta-aspartyl-AMP. This rescue experiment is the second example in which the function of a critical arginine residue that has been substituted by mutagenesis is restored by GdmHCl. Mutation of Thr-322 and Thr-323 also produces enzymes with altered kinetic properties, suggesting that these threonines are involved in aspartate binding and/or stabilization of intermediates en route to beta-aspartyl-AMP. These experiments are the first to identify residues outside of the N-terminal glutamine amide transfer domain that have any functional role in asparagine synthesis.


Subject(s)
Adenosine Monophosphate/analogs & derivatives , Asparagine/biosynthesis , Aspartate-Ammonia Ligase/chemistry , Aspartic Acid/analogs & derivatives , Adenosine Monophosphate/metabolism , Amino Acid Sequence , Arginine , Aspartate-Ammonia Ligase/genetics , Aspartic Acid/metabolism , Escherichia coli , Glutamine/metabolism , Kinetics , Models, Molecular , Molecular Sequence Data , Mutagenesis , Mutagenesis, Site-Directed , Sequence Alignment , Software , Structure-Activity Relationship , Threonine
13.
J Med Chem ; 39(12): 2367-78, 1996 Jun 07.
Article in English | MEDLINE | ID: mdl-8691431

ABSTRACT

Novel inhibitors of asparagine synthetase, that will lower circulating levels of blood asparagine, have considerable potential in developing new protocols for the treatment of acute lymphoblastic leukemia. We now report the indirect characterization of the aspartate binding site of Escherichia coli asparagine synthetase B (AS-B) using a number of stereochemically, and conformationally, defined aspartic acid analogs. Two compounds, prepared using novel reaction conditions for the stereospecific beta-functionalization of aspartic acid diesters, have been found to be competitive inhibitors with respect to aspartate in kinetic studies on AS-B. Chemical modification experiments employing [(fluorosulfonyl)benzoyl]adenosine (FSBA), an ATP analog, demonstrate that both inhibitors bind to the aspartate binding site of AS-B. Our results reveal that large steric alterations in the substrate are not tolerated by the enzyme, consistent with the failure of previous efforts to develop AS inhibitors using random screening approaches, and that all of the ionizable groups are placed in close proximity in the bound conformation of aspartate.


Subject(s)
Antineoplastic Agents/chemistry , Aspartate-Ammonia Ligase/chemistry , Aspartic Acid/metabolism , Bacterial Proteins/chemistry , Enzyme Inhibitors/pharmacology , Escherichia coli/enzymology , Isoenzymes/chemistry , Alkylation , Asparagine/biosynthesis , Aspartate-Ammonia Ligase/antagonists & inhibitors , Aspartate-Ammonia Ligase/metabolism , Aspartic Acid/analogs & derivatives , Bacterial Proteins/antagonists & inhibitors , Bacterial Proteins/metabolism , Binding Sites , Drug Design , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Glutamine/metabolism , Humans , Isoenzymes/antagonists & inhibitors , Isoenzymes/metabolism , Kinetics , Molecular Conformation , Molecular Structure , Neoplasm Proteins/antagonists & inhibitors , Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Precursor Cell Lymphoblastic Leukemia-Lymphoma/enzymology , Protein Binding , Stereoisomerism , Structure-Activity Relationship , Substrate Specificity
14.
Biochemistry ; 35(9): 3024-30, 1996 Mar 05.
Article in English | MEDLINE | ID: mdl-8608141

ABSTRACT

In experiments aimed at determining the mechanism of nitrogen transfer in purF amidotransferase enzymes, 13C and 15N kinetic isotope effects have been measured for both of the glutamine-dependent activities of Escherichia coli asparagine synthetase B (AS-B). For the glutaminase reaction catalyzed by AS-B at pH 8.0, substitution heavy atom labels in the side chain amide of the substrate yields observed values of 1.0245 and 1.0095 for the amide carbon and amide nitrogen isotope effects, respectively. In the glutamine-dependent synthesis of asparagine at pH 8.0, the amide carbon and amide nitrogen isotope effects have values of 1.0231 and 1.0222, respectively. We interpret these results to mean that nitrogen transfer does not proceed by the formation of free ammonia in the active site of the enzyme and probably involves a series of intermediates in which glutamine becomes covalently attached to aspartate. While a number of mechanisms are consistent with the observed isotope effects, a likely reaction pathway involves reaction of an oxyanion with beta-aspartyl-AMP. This yields an intermediate in which C-N bond cleavage gives an acylthioenzyme and a second tetrahedral intermediate. Loss of AMP from the latter gives asparagine. An alternate reaction mechanism in which asparagine is generated from an imide intermediate also appears consistent with the observed kinetic isotope effects.


Subject(s)
Aspartate-Ammonia Ligase/metabolism , Escherichia coli/enzymology , Glutaminase/metabolism , Aspartic Acid/metabolism , Carbon Isotopes , Genes, Bacterial , Glutamine/metabolism , Isotope Labeling , Kinetics , Models, Chemical , Nitrogen Isotopes , Substrate Specificity
15.
Biochemistry ; 35(9): 3031-7, 1996 Mar 05.
Article in English | MEDLINE | ID: mdl-8608142

ABSTRACT

Escherichia coli asparagine synthetase B (AS-B) catalyzes the synthesis of asparagine from aspartic acid and glutamine in an ATP-dependent reaction. The ability of this enzyme to employ hydroxylamine and L-glutamic acid gamma-monohydroxamate (LGH) as alternative substrates in place of ammonia and L-glutamine, respectively, has been investigated. The enzyme is able to function as an amidohydrolase, liberating hydroxylamine from LGH with high catalytic efficiency, as measured by k(cat)/K(M). In addition, the kinetic parameters determined for hydroxylamine in AS-B synthetase activity are very similar to those of ammonia. Nitrogen transfer from LGH to yield aspartic acid beta-monohydroxamate is also catalyzed by AS-B. While such an observation has been made for a few members of the trpG amidotransferase family, our results appear to be the first demonstration that nitrogen transfer can occur from glutamine analogs in a purF amidotransferase. However, k(cat)/K(M) for the ATP-dependent transfer of hydroxylamine from LGH to aspartic acid is reduced 3-fold relative to that for glutamine-dependent asparagine synthesis. Further, the AS-B mutant in which asparagine is replaced by alanine (N74A) can also use hydroxylamine as an alternate substrate to ammonia and catalyze the hydrolysis of LGH. The catalytic efficiencies (k(cat)/K(M)) of nitrogen transfer from LGH and L-glutamine to beta-aspartyl-AMP are almost identical for the N74A AS-B mutant. These observations support the proposal that Asn-74 plays a role in catalyzing glutamine-dependent nitrogen transfer. We interpret our kinetic data as further evidence against ammonia-mediated nitrogen transfer from glutamine in the purF amidotransferase AS-B. These results are consistent with two alternate chemical mechanisms that have been proposed for this reaction [Boehlein, S. K., Richards, N. G. J., Walworth, E. S., & Schuster, S. M. (1994) J. Biol. Chem. 269, 26789-26795].


Subject(s)
Aspartate-Ammonia Ligase/metabolism , Enzyme Inhibitors/metabolism , Escherichia coli/enzymology , Glutamates/metabolism , Hydroxamic Acids/metabolism , Hydroxylamines/metabolism , Aspartate Aminotransferases/antagonists & inhibitors , Hydroxylamine , Isoenzymes/metabolism , Kinetics , Models, Chemical , Plasmids , Recombinant Proteins/metabolism , Substrate Specificity
16.
J Biol Chem ; 270(36): 21062-7, 1995 Sep 08.
Article in English | MEDLINE | ID: mdl-7673134

ABSTRACT

Two proline mimetics, the enantiomers of 2-aza-bicyclo[2,2,1]heptane-3-carboxylic acid, have been incorporated in place of Pro30 into synthetic peptides based on the B-loop beta-sheet sequence of human transforming growth factor-alpha (TGF-alpha) (residues Cys21-Cys32). The peptides were further modified by inclusion of an N-terminal phenylalanine and constrained by formation of an intramolecular disulfide bond. While no mitogenic response was observed in the parental NR6 cell line, the peptides stimulated DNA synthesis in NR6/HER cells (NR6 fibroblasts transfected with the human epidermal growth factor receptor). Induction of DNA synthesis was dose dependent, with EC50 values in the range 130-330 microM; in the presence of low doses of TGF-alpha, the mitogenic effect of the peptides was additive, up to the plateau response achieved by maximal doses of TGF-alpha alone. These effects are consistent with the peptides acting via the same mechanism as TGF-alpha. Analysis of the structure of the peptides by NMR indicated that the presence of the mimetics significantly increased the propensity of the peptidyl-proline bond to adopt the cis conformation. These data confirm the role of the beta-sheet in receptor activation, and emphasize the importance of presentation of peptides in an appropriate conformation for recognition.


Subject(s)
Bridged Bicyclo Compounds/chemistry , Peptides/pharmacology , Proline/chemistry , Transforming Growth Factor alpha/pharmacology , Amino Acid Sequence , Animals , Cell Division/drug effects , Cell Line , Humans , Magnetic Resonance Spectroscopy , Mice , Molecular Mimicry , Molecular Sequence Data , Peptides/chemistry , Protein Conformation , Stereoisomerism , Transforming Growth Factor alpha/chemistry
17.
J Biol Chem ; 269(43): 26789-95, 1994 Oct 28.
Article in English | MEDLINE | ID: mdl-7929415

ABSTRACT

Although Arg-30, Asn-74, and Asn-79 appear totally conserved throughout the purF glutamine-dependent amidotransferases, their potential roles in catalysis and binding remain unexplored for any member of the enzyme family. Here we report the overexpression, purification, and kinetic characterization of a series of AS-B mutants which allow an examination of the functional roles of these 3 residues in glutamine-dependent nitrogen transfer. While Asn-79 appears to possess no catalytic function in AS-B, site-directed mutagenesis of Asn-74 has implicated this residue as playing a role in catalysis of nitrogen transfer from glutamine. The kinetic properties of the Asn-74 AS-B mutant enzymes appear consistent with both ammonia-mediated nitrogen transfer and two apparently novel mechanistic suggestions for this reaction involving either an oxyanion or imide intermediate (Richards, N. G. J., and Schuster, S. M. (1992) FEBS Lett. 313, 98-102). We also demonstrate that replacement of Arg-30 by an alanine residue yields an AS-B mutant for which the apparent Km for glutamine is increased in the glutamine-dependent synthesis of asparagine. In addition, ATP-dependent stimulation of the glutaminase activity of AS-B is modified or completely eliminated when Arg-30 is replaced by other amino acids. The latter observation may indicate the existence of a molecular switch involving Arg-30 which coordinates the two half-reactions catalyzed by the glutamine-dependent amidotransferases and synthetase domains of cellular AS-B.


Subject(s)
Asparagine/biosynthesis , Aspartate-Ammonia Ligase/metabolism , Escherichia coli/enzymology , Glutamine/metabolism , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Ammonia/metabolism , Arginine/genetics , Arginine/metabolism , Asparagine/genetics , Asparagine/metabolism , Aspartate-Ammonia Ligase/genetics , Base Sequence , Catalysis , Escherichia coli/genetics , Kinetics , Models, Chemical , Molecular Sequence Data , Mutagenesis, Site-Directed , Nitrogen/metabolism , Nucleotides/pharmacology , Sequence Homology, Amino Acid , Structure-Activity Relationship , Transaminases/genetics , Transaminases/metabolism
18.
J Biol Chem ; 269(10): 7450-7, 1994 Mar 11.
Article in English | MEDLINE | ID: mdl-7907328

ABSTRACT

The mechanism of nitrogen transfer in glutamine-dependent amidotransferases remains to be unambiguously established. We now report the overexpression, purification, and kinetic characterization of both the glutamine- and ammonia-dependent activities of Escherichia coli asparagine synthetase B (AS-B) and a series of mutants. In common with other members of the purF family of amidotransferases, the recombinant enzyme possesses an NH2-terminal cysteine residue. Replacement of Cys-1 by either alanine or serine results in a loss of glutaminase and glutamine-dependent activity, without out any significant effect upon ammonia-dependent asparagine synthesis. As previously observed for human AS (Sheng, S., Moraga-Amador, D., Van Heeke, G., Allison, R. D., Richards, N. G. J., and Schuster, S. M. (1993) J. Biol. Chem. 268, 16771-16780), glutamine is an inhibitor of the ammonia-dependent reaction catalyzed by both the Cys-1-->Ala (C1A) and Cys-1-->Ser (C1S) mutants of AS-B. In the case of C1A, the inhibition pattern suggests that an abortive complex is formed. This is consistent with a recent proposal implicating the formation of an imide intermediate in the nitrogen transfer reaction (Richards, N. G. J., and Schuster, S. M. (1992) FEBS Lett. 313, 98-102). In contrast, glutamine appears to be only a competitive inhibitor of the ammonia-dependent activity of C1S. Cys-1 does not appear to be required for glutamine binding. Replacement of Asp-33 by either asparagine or glutamic acid has little effect on the kinetic properties of the mutant enzymes when compared to wild-type AS-B. Cys-1 and Asp-33 are cognate to residues Cys-1 and Asp-29 in glutamine phosphoribosylpyrophosphate amidotransferase which have been proposed to be members of a catalytic triad responsible for mediating nitrogen transfer in this enzyme (Mei, B., and Zalkin, H. (1989) J. Biol. Chem. 264, 16613-16619). In the case of AS-B, although Cys-1 is essential for glutamine-dependent activity, Asp-33 does not appear to participate in mediating nitrogen transfer. In an effort to locate other residues which might form part of a "catalytic triad" in the glutamine amidotransferase domain of AS-B, we have expressed and characterized mutant proteins in which His-29 and His-80, which are conserved within the glutamine amidotransferase domain of purF amidotransferases, are replaced by alanine (H29A and H80A).(ABSTRACT TRUNCATED AT 400 WORDS)


Subject(s)
Aspartate-Ammonia Ligase/metabolism , Escherichia coli/enzymology , Glutamine/metabolism , Nitrogen/metabolism , Amino Acid Sequence , Animals , Asparagine/genetics , Aspartate-Ammonia Ligase/genetics , Aspartate-Ammonia Ligase/isolation & purification , Base Sequence , Catalysis , Cloning, Molecular , Cysteine/genetics , Humans , Kinetics , Molecular Sequence Data , Oligodeoxyribonucleotides , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid
19.
J Biol Chem ; 268(22): 16771-80, 1993 Aug 05.
Article in English | MEDLINE | ID: mdl-8102140

ABSTRACT

Cys-1 mutants of recombinant human asparagine synthetase were constructed and their ability to catalyze the glutamine-dependent nitrogen transfer reaction required for asparagine biosynthesis was determined. In agreement with previous work, altering Cys-1 to either Ala or Ser eliminated the glutamine-dependent activity while only minimally affecting the kinetic properties of the ammonia-dependent reaction. A lack of glutaminase activity in these mutants also allowed examination of glutamine binding in studies of the ability of glutamine to inhibit the ammonia-dependent production of asparagine. In both mutants, analysis of the observed kinetics indicated that glutamine inhibited ammonia-dependent asparagine synthesis through the formation of an abortive complex. This unanticipated observation suggests that the commonly accepted mechanism for nitrogen transfer from the primary amide of glutamine to aspartic acid in asparagine synthetase may have to be re-examined. A novel mechanistic proposal which is consistent with the formation of an abortive complex in the two Cys-1 mutants is presented.


Subject(s)
Ammonia/metabolism , Aspartate-Ammonia Ligase/metabolism , Cysteine/metabolism , Glutamine/pharmacology , Aspartate-Ammonia Ligase/antagonists & inhibitors , Aspartate-Ammonia Ligase/genetics , Base Sequence , Cloning, Molecular , Cysteine/genetics , DNA , Glutamine/metabolism , Humans , Hydrogen-Ion Concentration , Immunoblotting , Kinetics , Molecular Sequence Data , Molecular Structure , Mutagenesis, Site-Directed , Substrate Specificity
20.
Biopolymers ; 33(6): 971-84, 1993 Jun.
Article in English | MEDLINE | ID: mdl-8318669

ABSTRACT

The observation that short, linear alanine-based polypeptides form stable alpha-helices in aqueous solution has allowed the development of well-defined experimental systems with which to study the influence of amino acid sequence upon the stability of secondary structure. We have performed detailed conformational searches upon six alanine-based peptides in order to rationalize the observed variation in the alpha-helical stability in terms of side-chain-backbone and side-chain-side-chain interactions. Although a simple, gas-phase, potential model was used to obtain the conformational energies for these peptides, good agreement was obtained with experiment regarding their relative alpha-helical stabilities. Our calculations clearly indicate that valine, isoleucine, and phenylalanine residues should destabilize the alpha-helical conformation when included within alanine-based peptides because of energetically unfavorable side-chain-backbone interactions, which tend to result in the formation of regions of 3(10)-helix. In the case of valine, the destabilization most probably arises from entropic effects as the isopropyl side chain can assume more orientations in the 3(10)-helical form of the peptide. A detailed examination of very short-range interactions in these peptides has also indicated that an interaction, involving fewer than five consecutive residues, whose stabilizing effect reinforces that of the (i, i + 4) hydrogen bond may be the basis of the requirement for increased nucleation (sigma) and propagation parameters (s) required by Zimm-Bragg theory to predict the alpha-helical content for compounds in this class of short peptides. Our calculations complement recent work using modified Zimm-Bragg and Lifson-Roig theories of the helix-coil transition, and are consistent with molecular dynamics simulations upon linear peptides in aqueous solution.


Subject(s)
Alanine/chemistry , Peptides/chemistry , Protein Structure, Secondary , Amino Acid Sequence , Models, Molecular , Molecular Sequence Data
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