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1.
Biochemistry ; 40(51): 15456-63, 2001 Dec 25.
Article in English | MEDLINE | ID: mdl-11747420

ABSTRACT

The Sir2 enzyme family is responsible for a newly classified chemical reaction, NAD(+)-dependent protein deacetylation. New peptide substrates, the reaction mechanism, and the products of the acetyl transfer to NAD(+) are described for SIR2. The final products of SIR2 reactions are the deacetylated peptide and the 2' and 3' regioisomers of O-acetyl ADP ribose (AADPR), formed through an alpha-1'-acetyl ADP ribose intermediate and intramolecular transesterification reactions (2' --> 3'). The regioisomers, their anomeric forms, the interconversion rates, and the reaction equilibria were characterized by NMR, HPLC, 18O exchange, and MS methods. The mechanism of acetyl transfer to NAD(+) includes (1) ADP ribosylation of the peptide acyl oxygen to form a high-energy O-alkyl amidate intermediate, (2) attack of the 2'-OH group on the amidate to form a 1',2'-acyloxonium species, (3) hydrolysis to 2'-AADPR by the attack of water on the carbonyl carbon, and (4) an SIR2-independent transesterification equilibrating the 2'- and 3'-AADPRs. This mechanism is unprecedented in ADP-ribosyl transferase enzymology. The 2'- and 3'-AADPR products are candidate molecules for SIR2-initiated signaling pathways.


Subject(s)
Adenosine Diphosphate Ribose/analogs & derivatives , Adenosine Diphosphate Ribose/chemical synthesis , Gene Silencing , Histone Deacetylases/chemistry , NAD/chemistry , Silent Information Regulator Proteins, Saccharomyces cerevisiae , Trans-Activators/chemistry , Adenosine Diphosphate Ribose/chemistry , Adenosine Diphosphate Ribose/metabolism , Amino Acid Sequence , Arabinose/chemistry , Archaeal Proteins/chemistry , Archaeal Proteins/genetics , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Deuterium Oxide/metabolism , Enzyme Inhibitors/chemistry , Fungal Proteins/chemistry , Fungal Proteins/genetics , Histone Deacetylase Inhibitors , Histone Deacetylases/genetics , Histone Deacetylases/metabolism , Humans , Isomerism , Kinetics , Molecular Sequence Data , NAD/metabolism , Nuclear Magnetic Resonance, Biomolecular , O-Acetyl-ADP-Ribose , Oxygen Isotopes/metabolism , Sirtuin 1 , Sirtuin 2 , Sirtuins , Substrate Specificity , Trans-Activators/antagonists & inhibitors , Trans-Activators/genetics , Trans-Activators/metabolism
2.
Curr Opin Struct Biol ; 11(6): 657-65, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11751045

ABSTRACT

Atomic excursions of reactants in enzymatic catalytic sites can be estimated from high-resolution crystal structures of enzyme complexes with substrates, transition state analog inhibitors and products. Transition state structures, defined from kinetic isotope effect studies, are compared to crystallographic structures to validate the properties of the transition state analog. Atomic excursions in enzymatic catalytic sites can differ from those in solution and define the role of the enzymatic catalyst in directing atomic motion.


Subject(s)
DNA Glycosylases , Enzymes/metabolism , Animals , Catalytic Domain , Crystallography, X-Ray , Cytidine Deaminase/chemistry , Cytidine Deaminase/metabolism , Enzyme Inhibitors , Enzymes/chemistry , Humans , Motion , N-Glycosyl Hydrolases/chemistry , N-Glycosyl Hydrolases/metabolism , Protein Conformation , Protein Tyrosine Phosphatases/chemistry , Protein Tyrosine Phosphatases/metabolism , Purine-Nucleoside Phosphorylase/chemistry , Purine-Nucleoside Phosphorylase/metabolism , Ricin/chemistry , Ricin/metabolism , Uracil-DNA Glycosidase
3.
Curr Opin Chem Biol ; 5(5): 556-63, 2001 Oct.
Article in English | MEDLINE | ID: mdl-11578929

ABSTRACT

Experimental analysis of enzymatic transition states by kinetic isotope effect methods has established geometric variation in related transition state structures. Differences are apparent in development of the reaction coordinate, in solvolytic transition states relative to those in enzymatic catalytic sites, in the stereochemistry of related substrates at the transition state, and in reactions catalyzed by related enzymes.


Subject(s)
Enzymes/metabolism , Catalysis , DNA Glycosylases , Humans , Models, Chemical , N-Glycosyl Hydrolases/metabolism , Protein Conformation , Proteins/chemistry
4.
Laryngoscope ; 111(9): 1533-44, 2001 Sep.
Article in English | MEDLINE | ID: mdl-11568602

ABSTRACT

OBJECTIVE: The objective of this study was to evaluate the oncological outcome and complication rate following surgical treatment of nasopharyngeal salivary gland malignancy. STUDY DESIGN: Retrospective case review at tertiary care skull base center. METHODS: Pertinent medical records were reviewed from 23 patients presenting with minor salivary gland malignancy. Clinical presentation, prior treatment, histological type and grade, clinical stage, details of surgical treatment, and postoperative adjuvant radiation therapy were studied. Survival and recurrence data were analyzed using the Kaplan-Meier and Cox proportional hazards methods. RESULTS: Histological types included 11 adenoid cystic carcinomas, 8 mucoepidermoid carcinomas, and 4 cases of adenocarcinoma not otherwise specified. All patients underwent primary surgical resection, and the lateral infratemporal middle fossa approach was used in 20 patients. Prior radiation therapy had been administered in 6 patients who presented for treatment of recurrent disease, and the remaining 17 patients underwent planned postoperative radiation therapy. Elective neck dissection was undertaken in 15 patients, and occult neck disease was present in 47%. Disease specific survival was 67% at 5 years and 48% at 10 years. High-grade tumors had a significantly poorer outcome (P =.035) with a relative risk of 4.6 compared with low-grade disease. Local control was seen to be 77% at 5 years. CONCLUSIONS: Planned combined surgery and radiation therapy achieves survival outcomes and recurrence rates in nasopharyngeal salivary gland malignancy comparable to results reported using the same treatment for minor salivary gland tumors cancer originating elsewhere in the head and neck. Because of the high rate of occult neck metastases, we recommend elective neck dissection as part of the surgical treatment with this disease entity. The lateral infratemporal middle fossa approach provides safe and adequate access to resect the vast majority of these tumors with acceptable complication rates. A reliable form of vascularized reconstruction is necessary to prevent serious postoperative complications, and we currently prefer the gastro-omental free flap.


Subject(s)
Adenocarcinoma/surgery , Carcinoma, Adenoid Cystic/surgery , Carcinoma, Mucoepidermoid/surgery , Nasopharyngeal Neoplasms/surgery , Neoplasm Recurrence, Local , Salivary Gland Neoplasms/surgery , Actuarial Analysis , Adenocarcinoma/complications , Adenocarcinoma/mortality , Adenocarcinoma/pathology , Adolescent , Adult , Aged , Carcinoma, Adenoid Cystic/complications , Carcinoma, Adenoid Cystic/mortality , Carcinoma, Adenoid Cystic/pathology , Carcinoma, Mucoepidermoid/complications , Carcinoma, Mucoepidermoid/mortality , Carcinoma, Mucoepidermoid/pathology , Craniotomy/methods , Female , Humans , Magnetic Resonance Imaging , Male , Middle Aged , Nasopharyngeal Neoplasms/complications , Nasopharyngeal Neoplasms/mortality , Nasopharyngeal Neoplasms/pathology , Neck Dissection , Neoplasm Recurrence, Local/mortality , Neoplasm Recurrence, Local/pathology , Neoplasm Staging/methods , Patient Selection , Proportional Hazards Models , Radiotherapy, Adjuvant , Retrospective Studies , Risk Factors , Salivary Gland Neoplasms/complications , Salivary Gland Neoplasms/mortality , Salivary Gland Neoplasms/pathology , Surgical Flaps , Survival Analysis , Treatment Outcome
5.
Biochemistry ; 40(36): 10800-9, 2001 Sep 11.
Article in English | MEDLINE | ID: mdl-11535055

ABSTRACT

Adenine phosphoribosyltransferase (APRTase) is a widely distributed enzyme, and its deficiency in humans causes the accumulation of 2,8-dihydroxyadenine. It is the sole catalyst for adenine recycling in most eukaryotes. The most commonly expressed APRTase has subunits of approximately 187 amino acids, but the only crystal structure is from Leishmania donovani, which expresses a long form of the enzyme with 237 residues. Saccharomyces cerevisiae APRTase was selected as a representative of the short APRTases, and the structure of the apo-enzyme and sulfate bound forms were solved to 1.5 and 1.75 A, respectively. Yeast APRTase is a dimeric molecule, and each subunit is composed of a central five-stranded beta-sheet surrounded by five alpha-helices, a structural theme found in all known purine phosphoribosyltransferases. The structures reveal several important features of APRTase function: (i) sulfate ions bound at the 5'-phosphate and pyrophosphate binding sites; (ii) a nonproline cis peptide bond (Glu67-Ser68) at the pyrophosphate binding site in both apo-enzyme and sulfate-bound forms; and (iii) a catalytic loop that is open and ordered in the apo-enzyme but open and disordered in the sulfate-bound form. Alignment of conserved amino acids in short-APRTases from 33 species reveals 13 invariant and 15 highly conserved residues present in hinges, catalytic site loops, and the catalytic pocket. Mutagenesis of conserved residues in the catalytic loop, subunit interface, and phosphoribosylpyrophosphate binding site indicates critical roles for the tip of the catalytic loop (Glu106) and a catalytic site residue Arg69, respectively. Mutation of one loop residue (Tyr103Phe) increases k(cat) by 4-fold, implicating altered dynamics for the catalytic site loop.


Subject(s)
Adenine Phosphoribosyltransferase/chemistry , Adenine Phosphoribosyltransferase/metabolism , Saccharomyces cerevisiae/enzymology , Adenine Phosphoribosyltransferase/genetics , Amino Acid Sequence , Amino Acid Substitution , Animals , Apoenzymes/chemistry , Apoenzymes/metabolism , Bacteria/enzymology , Binding Sites , Cloning, Molecular , Dimerization , Drosophila/enzymology , Humans , Leishmania donovani/enzymology , Mice , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Protein Structure, Secondary , Protein Subunits , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/genetics , Sequence Alignment , Sequence Homology, Amino Acid , Sulfates/metabolism
6.
J Org Chem ; 66(17): 5723-30, 2001 Aug 24.
Article in English | MEDLINE | ID: mdl-11511245

ABSTRACT

Means have been developed for the synthesis and addition of 9-deaza-9-lithiopurine derivatives to the carbohydrate-derived cyclic imine 6 in facile convergent syntheses of biologically active aza-C-nucleosides.


Subject(s)
Imines/chemistry , Lithium/chemistry , Purines/chemistry , Pyrimidine Nucleosides/chemical synthesis , Pyrimidinones/chemical synthesis , Pyrroles/chemical synthesis
7.
Biochemistry ; 40(27): 8043-54, 2001 Jul 10.
Article in English | MEDLINE | ID: mdl-11434773

ABSTRACT

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.


Subject(s)
Enzyme Inhibitors/chemistry , Hypoxanthine Phosphoribosyltransferase/antagonists & inhibitors , Hypoxanthine Phosphoribosyltransferase/chemistry , Pyrimidinones/chemistry , Pyrroles/chemistry , Amino Acid Sequence , Binding Sites , Catalytic Domain , Chromatography, High Pressure Liquid , Deuterium/metabolism , Diphosphates/chemistry , Humans , Hypoxanthine Phosphoribosyltransferase/metabolism , Isoleucine/chemistry , Leucine/chemistry , Macromolecular Substances , Magnesium/chemistry , Magnesium Compounds/chemistry , Mass Spectrometry , Molecular Sequence Data , Pepsin A/metabolism , Peptide Fragments/antagonists & inhibitors , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Phenylalanine/chemistry , Protons
8.
Biochemistry ; 40(28): 8196-203, 2001 Jul 27.
Article in English | MEDLINE | ID: mdl-11444965

ABSTRACT

Purine salvage pathways are predicted to be present from the genome sequence of Mycobacterium tuberculosis. The M. tuberculosis deoD gene encodes a presumptive purine nucleoside phosphorylase (PNP). The gene was cloned, expressed, purified, and found to exhibit PNP activity. Purified M. tuberculosis PNP is trimeric, similar to mammalian PNP's but unlike the hexameric Escherichia coli enzyme. Immucillin-H is a rationally designed analogue of the transition state that has been shown to be a potent inhibitor of mammalian PNP's. This inhibitor also exhibits slow-onset inhibition of M. tuberculosis PNP with a rapid, reversible inhibitor binding (K(i) of 2.2 nM) followed by an overall dissociation constant (K(i)) of 28 pM, yielding a K(m)/K(i) value of 10(6). Time-dependent tight binding of the inhibitor occurs with a rate of 0.1 s(-)(1), while relaxation of the complex is slower at 1.4 x 10(-)(3) s(-)(1). The pH dependence of the K(i) value of immucillin-H to the M. tuberculosis PNP suggests that the inhibitor binds as the neutral, unprotonated form that is subsequently protonated to generate the tight-binding species. The M. tuberculosis enzyme demonstrates independent and equivalent binding of immucilin-H at each of the three catalytic sites, unlike mammalian PNP. Analysis of the components of immucillin-H confirms that the inhibition gains most of its binding energy from the 9-deazahypoxanthine group (K(is) of 0.39 microM) while the 1,4-dideoxy-1,4-iminoribitol binds weakly (K(is) of 2.9 mM). Double-inhibition studies demonstrate antagonistic binding of 9-deazahypoxanthine and iminoribitol (beta = 13). However, the covalent attachment of these two components in immucillin-H increases equilibrium binding affinity by a factor of >14 000 (28 pM vs 0.39 microM) compared to 9-deazahypoxanthine alone, and by a factor of >10(8) compared to iminoribitol alone (28 pM vs 2.9 mM), from initial velocity measurements. The structural basis for M. tuberculosis PNP inhibition by immucillin-H and by its component parts is reported in the following paper [Shi, W., Basso, L. A., Santos, D. S., Tyler, P. C., Furneaux, R. H., Blanchard, J. S., Almo, S. C., and Schramm, V. L. (2001) Biochemistry 40, 8204-8215].


Subject(s)
Enzyme Inhibitors/chemistry , Mycobacterium tuberculosis/enzymology , Purine-Nucleoside Phosphorylase/antagonists & inhibitors , Purine-Nucleoside Phosphorylase/chemistry , Pyrimidinones/chemistry , Pyrroles/chemistry , Binding, Competitive , Catalysis , Cloning, Molecular , Gene Expression Regulation, Bacterial , Hydrogen-Ion Concentration , Kinetics , Molecular Weight , Purine Nucleosides , Purine-Nucleoside Phosphorylase/biosynthesis , Purine-Nucleoside Phosphorylase/isolation & purification , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification
9.
Biochemistry ; 40(28): 8204-15, 2001 Jul 27.
Article in English | MEDLINE | ID: mdl-11444966

ABSTRACT

A structural genomics comparison of purine nucleoside phosphorylases (PNPs) indicated that the enzyme encoded by Mycobacterium tuberculosis (TB-PNP) resembles the mammalian trimeric structure rather than the bacterial hexameric PNPs. The crystal structure of M. tuberculosis PNP in complex with the transition-state analogue immucillin-H (ImmH) and inorganic phosphate was solved at 1.75 A resolution and confirms the trimeric structure. Binding of the inhibitor occurs independently at the three catalytic sites, unlike mammalian PNPs which demonstrate negative cooperativity in ImmH binding. Reduced subunit interface contacts for TB-PNP, compared to the mammalian enzymes, correlate with the loss of the cooperative inhibitor binding. Mammalian and TB-PNPs both exhibit slow-onset inhibition and picomolar dissociation constants for ImmH. The structure supports a catalytic mechanism of reactant destabilization by neighboring group electrostatic interactions, transition-state stabilization, and leaving group activation. Despite an overall amino acid sequence identity of 33% between bovine and TB-PNPs and almost complete conservation in active site residues, one catalytic site difference suggests a strategy for the design of transition-state analogues with specificity for TB-PNP. The structure of TB-PNP was also solved to 2.0 A with 9-deazahypoxanthine (9dHX), iminoribitol (IR), and PO(4) to reconstruct the ImmH complex with its separate components. One subunit of the trimer has 9dHX, IR, and PO(4) bound, while the remaining two subunits contain only 9dHX. In the filled subunit, 9dHX retains the contacts found in the ImmH complex. However, the region of IR that corresponds to the oxocarbenium ion is translocated in the direction of the reaction coordinate, and the nucleophilic phosphate rotates away from the IR group. Loose packing of the pieces of ImmH in the catalytic site establishes that covalent connectivity in ImmH is required to achieve the tightly bound complex.


Subject(s)
Enzyme Inhibitors/chemistry , Mycobacterium tuberculosis/enzymology , Purine-Nucleoside Phosphorylase/antagonists & inhibitors , Purine-Nucleoside Phosphorylase/chemistry , Pyrimidinones/chemistry , Pyrroles/chemistry , Actinomycetales/enzymology , Animals , Binding Sites , Catalysis , Cattle , Enzyme Stability , Escherichia coli/enzymology , Macromolecular Substances , Models, Molecular , Phosphates/chemistry , Protein Conformation , Purine Nucleosides
10.
J Am Chem Soc ; 123(7): 1327-36, 2001 Feb 21.
Article in English | MEDLINE | ID: mdl-11456704

ABSTRACT

Anomeric equilibrium isotope effects for dissolved sugars are required preludes to understanding isotope effects for these molecules bound to enzymes. This paper presents a full molecule study of the alpha- and beta-anomeric forms of D-glucopyranose in water using deuterium conformational equilibrium isotope effects (CEIE). Using 1D (13)C NMR, we have found deuterium isotope effects of 1.043 +/- 0.004, 1.027 +/- 0.005, 1.027 +/- 0.004, 1.001 +/- 0.003, 1.036 +/- 0.004, and 0.998 +/- 0.004 on the equilibrium constant, (H/D)K(beta/alpha), in [1-(2)H]-, [2-(2)H]-, [3-(2)H]-, [4-(2)H]-, [5-(2)H]-, and [6,6'-(2)H(2)]-labeled sugars, respectively. A computational study of the anomeric equilibrium in glucose using semiempirical and ab initio methods yields values that correlate well with experiment. Natural bond orbital (NBO) analysis of glucose and dihedral rotational equilibrium isotope effects in 2-propanol strongly imply a hyperconjugative mechanism for the isotope effects at H1 and H2. We conclude that the isotope effect at H1 is due to n(p) --> sigma* hyperconjugative transfer from O5 to the axial C1--H1 bond in beta-glucose, while this transfer makes no contribution to the isotope effect at H5. The isotope effect at H2 is due to rotational restriction of OH2 at 160 degrees in the alpha form and 60 degrees in the beta-sugar, with concomitant differences in n --> sigma* hyperconjugative transfer from O2 to CH2. The isotope effects on H3 and H5 result primarily from syn-diaxial steric repulsion between these and the axial anomeric hydroxyl oxygen in alpha-glucose. Therefore, intramolecular effects play an important role in isotopic perturbation of the anomeric equilibrium. The possible role of intermolecular effects is discussed in the context of recent molecular dynamics studies on aqueous glucose.


Subject(s)
Glucose/chemistry , Carbon Isotopes , Deuterium , Magnetic Resonance Spectroscopy , Molecular Conformation
11.
Biochemistry ; 40(23): 6845-51, 2001 Jun 12.
Article in English | MEDLINE | ID: mdl-11389598

ABSTRACT

Ricin toxin A-chain (RTA) is expressed by the castor bean plant and is among the most potent mammalian toxins. Upon activation in the cytosol, RTA depurinates a single adenine from position 4324 of rat 28S ribosomal RNA, causing inactivation of ribosomes by preventing the binding of elongation factors. Kinetic isotope effect studies have established that RTA operates via a D(N)*A(N) mechanism involving an oxacarbenium ion intermediate with bound adenine [Chen, X.-Y., Berti, P. J., and Schramm, V. L. (2000) J. Am. Chem. Soc. 122, 1609-1617]. On the basis of this information, stem-loop RNA molecules were chemically synthesized, incorporating structural features of the oxacarbenium ion-like transition state. A 10-base RNA stem-loop incorporating (1S)-1-(9-deazaadenin-9-yl)-1,4-dideoxy-1,4-imino-D-ribitol at the depurination site binds four times better (0.57 microM) than the 10-base RNA stem-loop with adenosine at the depurination site (2.2 microM). A 10-base RNA stem-loop with 1,2-dideoxyribitol [(2R,3S)-2-(hydroxymethyl)-3-hydroxytetrahydrofuran] at the depurination site binds with a Kd of 3.2 microM and tightens to 0.75 microM in the presence of 9-deazaadenine. A similar RNA stem-loop with 1,4-dideoxy-1,4-imino-D-ribitol at the depurination site binds with a K(d) of 1.3 microM and improves to 0.65 micro;M with 9-deazaadenine added. When (3S,4R)-4-hydroxy-3-(hydroxymethyl)pyrrolidine was incorporated at the depurination site of a 14-base RNA stem-loop, the Kd was 0.48 microM. Addition of 9-deazaadenine tightens the binding to 0.10 microM whereas added adenine increases the affinity to 12 nM. The results of this study are consistent with the unusual dissociative D(N)*A(N) mechanism determined for RTA. Knowledge of this intermediate has led to the design and synthesis of the highest affinity inhibitor reported for the catalytic site of RTA.


Subject(s)
Adenine/analogs & derivatives , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/metabolism , Ricin/antagonists & inhibitors , Ricin/chemistry , Adenine/chemistry , Adenine/metabolism , Amides/chemistry , Amides/metabolism , Animals , Binding, Competitive , Enzyme Inhibitors/chemical synthesis , Hydrolysis , Nucleic Acid Conformation , Nucleosides/chemical synthesis , Nucleosides/chemistry , Nucleosides/metabolism , Phosphoric Acids/chemistry , Phosphoric Acids/metabolism , RNA, Ribosomal, 28S/chemistry , RNA, Ribosomal, 28S/metabolism , Rats , Ricin/metabolism , Substrate Specificity
12.
Proc Natl Acad Sci U S A ; 98(8): 4593-8, 2001 Apr 10.
Article in English | MEDLINE | ID: mdl-11287638

ABSTRACT

Transition-state theory has led to the design of Immucillin-H (Imm-H), a picomolar inhibitor of purine nucleoside phosphorylase (PNP). In humans, PNP is the only route for degradation of deoxyguanosine, and genetic deficiency of this enzyme leads to profound T cell-mediated immunosuppression. This study reports the biological effects and mechanism of action of Imm-H on malignant T cell lines and on normal activated human peripheral T cells. Imm-H inhibits the growth of malignant T cell leukemia lines with the induction of apoptosis. Imm-H also inhibits activated normal human T cells after antigenic stimulation in vitro. However, Imm-H did not inhibit malignant B cells, colon cancer cell lines, or normal human nonstimulated T cells, demonstrating the selective activity of Imm-H. The effects on leukemia cells were mediated by the cellular phosphorylation of deoxyguanosine and the accumulation of dGTP, an inhibitor of ribonucleotide diphosphate reductase. Cells were protected from the toxic effects of Imm-H when deoxyguanosine was absent or when deoxycytidine was present. Guanosine incorporation into nucleic acids was selectively blocked by Imm-H with no effect on guanine, adenine, adenosine, or deoxycytidine incorporation. Imm-H may have clinical potential for treatment of human T cell leukemia and lymphoma and for other diseases characterized by abnormal activation of T lymphocytes. The design of Imm-H from an enzymatic transition-state analysis exemplifies a powerful approach for developing high-affinity enzyme inhibitors with pharmacologic activity.


Subject(s)
Enzyme Inhibitors/pharmacology , Purine-Nucleoside Phosphorylase/antagonists & inhibitors , Pyrimidinones/pharmacology , Pyrroles/pharmacology , T-Lymphocytes/drug effects , Apoptosis/drug effects , Cell Division/drug effects , Deoxyguanine Nucleotides/metabolism , Enzyme Inhibitors/toxicity , Humans , Purine Nucleosides , Pyrimidinones/toxicity , Pyrroles/toxicity , T-Lymphocytes/cytology , T-Lymphocytes/metabolism , Tumor Cells, Cultured
13.
Biochemistry ; 40(4): 853-60, 2001 Jan 30.
Article in English | MEDLINE | ID: mdl-11170405

ABSTRACT

Immucillin-H [ImmH; (1S)-1-(9-deazahypoxanthin-9-yl)-1,4-dideoxy-1,4-imino-D-ribitol] is a 23 pM inhibitor of bovine purine nucleoside phosphorylase (PNP) specifically designed as a transition state mimic [Miles, R. W., Tyler, P. C., Furneaux, R. H., Bagdassarian, C. K., and Schramm, V. L. (1998) Biochemistry 37, 8615-8621]. Cocrystals of PNP and the inhibitor are used to provide structural information for each step through the reaction coordinate of PNP. The X-ray crystal structure of free ImmH was solved at 0.9 A resolution, and a complex of PNP.ImmH.PO(4) was solved at 1.5 A resolution. These structures are compared to previously reported complexes of PNP with substrate and product analogues in the catalytic sites and with the experimentally determined transition state structure. Upon binding, ImmH is distorted to a conformation favoring ribosyl oxocarbenium ion formation. Ribosyl destabilization and transition state stabilization of the ribosyl oxocarbenium ion occur from neighboring group interactions with the phosphate anion and the 5'-hydroxyl of the ribosyl group. Leaving group activation of hypoxanthine involves hydrogen bonds to O6, N1, and N7 of the purine ring. Ordered water molecules provide a proton transfer bridge to O6 and N7 and permit reversible formation of these hydrogen bonds. Contacts between PNP and catalytic site ligands are shorter in the transition state analogue complex of PNP.ImmH.PO(4) than in the Michaelis complexes of PNP.inosine.SO(4) or PNP.hypoxanthine.ribose 1-PO(4). Reaction coordinate motion is dominated by translation of the carbon 1' of ribose between relatively fixed phosphate and purine groups. Purine and pyrimidine phosphoribosyltransferases and nucleoside N-ribosyl hydrolases appear to operate by a similar mechanism.


Subject(s)
Purine-Nucleoside Phosphorylase/chemistry , Animals , Binding Sites , Catalysis , Cattle , Crystallography, X-Ray , Deuterium/chemistry , Electron Transport , Enzyme Inhibitors/chemistry , Hydrolysis , Inosine/chemistry , Macromolecular Substances , Motion , Phosphates/chemistry , Protein Conformation , Purine Nucleosides , Purine-Nucleoside Phosphorylase/antagonists & inhibitors , Pyrimidinones/chemistry , Pyrroles/chemistry
14.
Biochemistry ; 40(1): 9-14, 2001 Jan 09.
Article in English | MEDLINE | ID: mdl-11141051

ABSTRACT

The mechanism of propagation of the radical center between the cofactor, substrate, and product in the adenosylcobalamin- (AdoCbl) dependent reaction of ethanolamine ammonia-lyase has been probed by pulsed electron nuclear double resonance (ENDOR) spectroscopy. The radical of S-2-aminopropanol, which appears in the steady state of the reaction, was used in ENDOR experiments to determine the nuclear spin transition frequencies of (2)H introduced from either deuterated substrate or deuterated coenzyme and of (13)C introduced into the ribosyl moiety of AdoCbl. A (2)H doublet (1.4 MHz splitting) was observed centered about the Larmor frequency of (2)H. Identical ENDOR frequencies were observed for (2)H irrespective of its mode of introduction into the complex. A (13)C doublet ENDOR signal was observed from samples prepared with [U-(13)C-ribosyl]-AdoCbl. The (13)C coupling tensor obtained from the ENDOR powder pattern shows that the (13)C has scalar as well as dipole-dipole coupling to the unpaired electron located at C1 of S-2-aminopropanol. The dipole-dipole coupling is consistent with a distance of 3.4+/-0.2 A between C1 of the radical and C5' of the labeled cofactor component. These results establish that the C5' carbon of the 5'-deoxyadenosyl radical moves approximately 7 A from its position as part of AdoCbl to a position where it is in contact with C1 of the substrate which lies approximately 12 A from the Co(2+) of cob(II)alamin. These findings are also consistent with the contention that 5'-deoxyadenosine is the sole mediator of hydrogen transfers in ethanolamine ammonia-lyase.


Subject(s)
Deoxyadenosines/chemistry , Ethanolamine Ammonia-Lyase/chemistry , Binding Sites , Carbon Isotopes , Cobamides/chemistry , Deuterium , Electron Spin Resonance Spectroscopy/methods , Free Radicals/chemistry , Propanolamines/chemistry , Substrate Specificity
15.
Protein Sci ; 9(9): 1660-8, 2000 Sep.
Article in English | MEDLINE | ID: mdl-11045613

ABSTRACT

The rate and extent of hydrogen/deuterium (H/D) exchange into purine nucleoside phosphorylase (PNP) was monitored by electrospray ionization mass spectrometry (ESI-MS) to probe protein conformational and dynamic changes induced by a substrate analogue, products, and a transition state analogue. The genetic deficiency of PNP in humans is associated with severe T-cell immunodeficiency, while B-cell immunity remains functional. Inhibitors of PNP have been proposed for treatment of T-cell leukemia, to suppress the graft-vs.-host response, or to counter type IV autoimmune diseases without destroying humoral immunity. Calf spleen PNP is a homotrimer of polypeptide chains with 284 amino residues, molecular weight 31,541. Immucillin-H inhibits PNP with a Kd of 23 pM when only one of the three catalytic sites is occupied. Deuterium exchange occurs at 167 slow-exchange sites in 2 h when no catalytic site ligands are present. The substrate analogue and product prevented H/D exchange at 10 of the sites. Immucillin-H protected 32 protons from exchange at full saturation. When one of the three subunits of the homotrimer is filled with immucillin-H, and 27 protons are protected from exchange in all three subunits. Deuterium incorporation in peptides from residues 132-152 decreased in all complexes of PNP. The rate and/or extent of deuterium incorporation in peptides from residues 29-49, 50-70, 81-98, and 112-124 decreased only in the complex with the transition state analogue. The peptide-specific H/D exchange demonstrates that (1) the enzyme is most compact in the complex with immucillin-H, and (2) filling a single catalytic site of the trimer reduces H/D exchange in the same peptides in adjacent subunits. The peptides most highly influenced by the inhibitor surround the catalytic site, providing evidence for reduced protein dynamic motion caused by the transition state analogue.


Subject(s)
Purine-Nucleoside Phosphorylase/metabolism , Pyrimidinones/metabolism , Pyrroles/metabolism , Amino Acid Sequence , Mass Spectrometry , Models, Molecular , Molecular Sequence Data , Protein Binding , Purine Nucleosides , Pyrimidinones/chemistry , Pyrroles/chemistry , Solvents
16.
Biochemistry ; 39(23): 6781-90, 2000 Jun 13.
Article in English | MEDLINE | ID: mdl-10841757

ABSTRACT

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.


Subject(s)
Giardia lamblia/enzymology , Hypoxanthine Phosphoribosyltransferase/chemistry , Animals , Binding Sites , Crystallography, X-Ray , Dimerization , Enzyme Inhibitors/chemistry , Models, Molecular , Molecular Conformation , Pyrimidinones/chemistry , Pyrroles/chemistry , Recombinant Proteins/chemistry
17.
Biochemistry ; 38(49): 16076-83, 1999 Dec 07.
Article in English | MEDLINE | ID: mdl-10587430

ABSTRACT

A computational method has been developed to predict inhibitor binding energy for untested inhibitor molecules. A neural network is trained from the electrostatic potential surfaces of known inhibitors and their binding energies. The algorithm is then able to predict, with high accuracy, the binding energy of unknown inhibitors. IU-nucleoside hydrolase from Crithidia fasciculata and the inhibitor molecules described previously [Miles, R. W. Tyler, P. C. Evans, G. Furneaux R. H., Parkin, D. W., and Schramm, V. L. (1999) Biochemistry 38, xxxx-xxxx] are used as the test system. Discrete points on the molecular electrostatic potential surface of inhibitor molecules are input to neural networks to identify the quantum mechanical features that contribute to binding. Feed-forward neural networks with back-propagation of error are trained to recognize the quantum mechanical electrostatic potential and geometry at the entire van der Waals surface of a group of training molecules and to predict the strength of interactions between the enzyme and novel inhibitors. The binding energies of unknown inhibitors were predicted, followed by experimental determination of K(i)() values. Predictions of K(i)() values using this theory are compared to other methods and are more robust in estimating inhibitory strength. The average deviation in estimating K(i)() values for 18 unknown inhibitor molecules, with 21 training molecules, is a factor of 5 x K(i)() over a range of 660 000 in K(i)() values for all molecules. The a posteriori accuracy of the predictions suggests the method will be effective as a guide for experimental inhibitor design.


Subject(s)
Crithidia fasciculata/enzymology , Enzyme Inhibitors/chemistry , N-Glycosyl Hydrolases/antagonists & inhibitors , N-Glycosyl Hydrolases/chemistry , Neural Networks, Computer , Ribonucleosides/chemistry , Animals , Binding Sites , Computer Simulation , Enzyme Inhibitors/metabolism , Hydrogen Bonding , Hydrolysis , N-Glycosyl Hydrolases/metabolism , Quantum Theory , Ribonucleosides/metabolism , Ribose/chemistry , Ribose/metabolism , Static Electricity , Structure-Activity Relationship , Surface Properties , Thermodynamics
19.
Biochemistry ; 38(40): 13147-54, 1999 Oct 05.
Article in English | MEDLINE | ID: mdl-10529186

ABSTRACT

Nucleoside N-ribohydrolases from protozoan parasites are targets for inhibitor design in these purine-auxotrophic organisms. Purine-specific and purine/pyrimidine-nonspecific nucleoside hydrolases have been reported. Iminoribitols that are 1-substituted with meta- and para-derivatized phenyl groups [(1S)-substituted 1, 4-dideoxy-1,4-imino-D-ribitols] are powerful inhibitors for the nonspecific nucleoside N-ribohydrolases, but are weak inhibitiors for purine-specific isozymes [Parkin, D. W., Limberg, G., Tyler, P. C., Furneaux, R. H., Chen, X.-Y., and Schramm, V. L. (1997) Biochemisty 36, 3528-3534]. Binding of these inhibitors to nonspecific nucleoside hydrolase occurs primarily via interaction with the iminoribitol, a ribooxocarbenium ion analogue of the transition state. Weaker interactions arise from hydrophobic interactions between the phenyl group and the purine/pyrimidine site. In contrast, the purine-specific enzymes obtain equal catalytic potential from leaving group activation and ribooxocarbenium ion formation. Knowledge of the reaction mechanisms and transition states for these enzymes has guided the design of isozyme-specific transition state analogue inhibitors. New synthetic efforts have produced novel inhibitors that incorporate features of the leaving group hydrogen-bonding sites while retaining the iminoribitol group. These compounds provide the first transition state analogue inhibitors for purine-specific nucleoside hydrolase. The most inhibitory 1-substituted iminoribitol heterocycle is a sub-nanomolar inhibitor for the purine-specific nucleoside hydrolase from Trypanosoma brucei brucei. Novel nanomolar inhibitors are also described for the nonspecific nucleoside hydrolase from Crithidia fasciculata. The compounds reported here are the most powerful iminoribitol inhibitors yet described for the nucleoside hydrolases.


Subject(s)
Enzyme Inhibitors/chemistry , N-Glycosyl Hydrolases/antagonists & inhibitors , Protozoan Proteins/antagonists & inhibitors , Ribitol/analogs & derivatives , Animals , Crithidia fasciculata/enzymology , Guanosine/chemistry , Hydrogen Bonding , Inosine/chemistry , Macromolecular Substances , N-Glycosyl Hydrolases/chemistry , Protozoan Proteins/chemistry , Purines/chemistry , Pyrimidinones/chemistry , Pyrroles/chemistry , Ribitol/chemistry , Ribose/chemistry , Structure-Activity Relationship , Substrate Specificity
20.
Biochemistry ; 38(31): 9872-80, 1999 Aug 03.
Article in English | MEDLINE | ID: mdl-10433693

ABSTRACT

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.


Subject(s)
Enzyme Inhibitors/chemistry , Pentosyltransferases/antagonists & inhibitors , Pentosyltransferases/chemistry , Plasmodium falciparum/enzymology , Pyrimidinones/chemistry , Pyrroles/chemistry , Animals , Catalytic Domain , Crystallography, X-Ray , Humans , Macromolecular Substances , Models, Molecular , Nuclear Magnetic Resonance, Biomolecular , Protein Conformation , Protein Folding , Protein Structure, Secondary , Protons , Purine Nucleosides
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