ABSTRACT
Tautomerase superfamily members have an amino-terminal proline and a beta-alpha-beta fold, and include 4-oxalocrotonate tautomerase (4-OT), 5-(carboxymethyl)-2-hydroxymuconate isomerase (CHMI), trans- and cis-3-chloroacrylic acid dehalogenase (CaaD and cis-CaaD, respectively), malonate semialdehyde decarboxylase (MSAD), and macrophage migration inhibitory factor (MIF), which exhibits a phenylpyruvate tautomerase (PPT) activity. Pro-1 is a base (4-OT, CHMI, the PPT activity of MIF) or an acid (CaaD, cis-CaaD, MSAD). Components of the catalytic machinery have been identified and mechanistic hypotheses formulated. Characterization of new homologues shows that these mechanisms are incomplete. 4-OT, CaaD, cis-CaaD, and MSAD also have promiscuous activities with a hydratase activity in CaaD, cis-CaaD, and MSAD, PPT activity in CaaD and cis-CaaD, and CaaD and cis-CaaD activities in 4-OT. The shared promiscuous activities provide evidence for divergent evolution from a common ancestor, give hints about mechanistic relationships, and implicate catalytic promiscuity in the emergence of new enzymes.
Subject(s)
Carbon-Carbon Double Bond Isomerases/genetics , Carboxy-Lyases/genetics , Evolution, Molecular , Hydrolases/genetics , Isomerases/genetics , Macrophage Migration-Inhibitory Factors/genetics , Models, Molecular , Protein Structure, Secondary/genetics , Carbon-Carbon Double Bond Isomerases/chemistry , Carboxy-Lyases/chemistry , Catalysis , Hydrolases/chemistry , Isomerases/chemistry , Macrophage Migration-Inhibitory Factors/chemistryABSTRACT
Chemically synthesized small interfering RNAs (siRNAs) are tools used for silencing the expression of a single gene. They are mainly employed in basic research applications, but may also have great potential in therapeutic applications. Longer double-stranded RNAs, such as Dicer-substrate 27mers, trigger gene silencing through the intrinsic RNAi pathway. The design of these Dicer-substrate 27mers has been optimized so they can be oriented by Dicer to consistently select the antisense (guide) strand after cleavage to shorter siRNAs, leading to predictable mRNA cleavage. In this paper we describe evidence that these Dicer-substrate 27mers produce more potent and sustained gene silencing for four genes when compared with synthetic 21mers that have the same guide-strand sequence. Furthermore, improved silencing by these 27mers is often more pronounced at lower concentrations.
Subject(s)
Gene Silencing , Ribonuclease III/metabolism , Base Sequence , Biotechnology , Cyclin-Dependent Kinase 2/genetics , DNA Primers/genetics , Genes, p53 , HeLa Cells , Humans , Models, Genetic , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-raf/genetics , RNA Interference , RNA, Small Interfering/chemical synthesis , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Substrate Specificity , Time Factors , TransfectionABSTRACT
BACKGROUND: Workers in hydroelectric plants appear to be readily sensitized to caddisfly allergens. This sensitization probably occurs de novo from occupational exposure. In some workers, sensitization occurs on a non-atopic background. Cytokine synthesis of IFN-gamma, IL-5 and IL-13 in atopic and non-atopic caddisfly-allergic workers was examined to determine if responses were similar or different. METHODS: Peripheral blood mononuclear cells were isolated from atopic caddisfly-allergic workers, non-atopic caddisfly-allergic workers and non-atopic caddisfly-exposed but non-allergic workers. Stimulation with caddisfly antigens was carried out and synthesis of IFN-gamma, IL-5 and IL-13 was determined by sandwich ELISA. RESULTS: Both caddisfly-allergic and non-allergic subjects responded to stimulation with caddisfly extract. The response in non-atopic caddisfly-non-allergic subjects was TH1 predominant, while that in atopic caddisfly-allergic subjects was TH2 predominant. The response in non-atopic caddisfly-allergic subjects was between that of the atopic caddisfly-allergic workers and the non-atopic caddisfly-non-allergic workers and the trend was to a TH2 response. Work-related symptoms were similarly intermediate between the atopic caddisfly-allergic and non-atopic caddisfly-non-allergic group. Differences were significant for IFN-gamma/IL-5 ratios but not IFN-gamma/IL-13 ratios for atopic and non-atopic caddisfly-allergic individuals, compared to non-atopic caddisfly-non-allergic workers. However, a linear relationship existed between IFN-gamma synthesis and IL-5 and IL-13 synthesis in non-atopic caddisfly-allergic workers but not in atopic caddisfly-allergic subjects. CONCLUSIONS: Caddisfly allergy in hydroelectric workers may be a useful model for the development of allergy to a previously unencountered allergen, and points to some interesting differences between atopic and non-atopic subjects who become sensitized to environmental allergens.
Subject(s)
Allergens/immunology , Conjunctivitis, Allergic/immunology , Cytokines/immunology , Dermatitis, Atopic/immunology , Insecta/immunology , Occupational Diseases/immunology , Power Plants , Animals , Cytokines/metabolism , Dose-Response Relationship, Immunologic , Humans , Insecta/metabolism , Occupational Exposure/adverse effects , Skin Tests , Th1 Cells/immunology , Th2 Cells/immunologyABSTRACT
The amino-terminal proline of 4-oxalocrotonate tautomerase (4-OT) functions as the general base catalyst in the enzyme-catalyzed isomerization of beta,gamma-unsaturated enones to their alpha,beta-isomers because of its unusually low pK(a) of 6.4 +/- 0.2, which is 3 units lower than that of the model compound, proline amide. Recent studies show that this abnormally low pK(a) is not due to the electrostatic effects of nearby cationic residues (Arg-11, Arg-39, and Arg-61) [Czerwinski, R. M., Harris, T. K., Johnson, Jr., W. H., Legler, P. M., Stivers, J. T., Mildvan, A. S., and Whitman, C. P. (1999) Biochemistry 38, 12358-12366]. Hence, it may result solely from a low local dielectric constant of 14.7 +/- 0.8 at the otherwise hydrophobic active site. Support for this mechanism comes from the study of mutants of the active site Phe-50, which is 5.8 A from Pro-1 and is one of 12 apolar residues within 9 A of Pro-1. Replacing Phe-50 with Tyr does not significantly alter k(cat) or K(m) and results in a pK(a) of 6.0 +/- 0.1 for Pro-1 as determined by (15)N NMR spectroscopy, comparable to that observed for wild type. (1)H-(15)N HSQC and 3D (1)H-(15)N NOESY HSQC spectra of the F50Y mutant demonstrate its conformation to be very similar to that of the wild-type enzyme. In the F50Y mutant, the pK(a) of Tyr-50 is increased by two units from that of a model compound N-acetyl-tyrosine amide to 12.2 +/- 0.3, as determined by UV and (1)H NMR titrations, yielding a local dielectric constant of 13.4 +/- 1.7, in agreement with the value of 13.7 +/- 0.3 determined from the decreased pK(a) of Pro-1 in this mutant. In the F50A mutant, the pK(a) of Pro-1 is 7.3 +/- 0.1 by (15)N NMR titration, comparable to the pK(a) of 7.6 +/- 0.2 found in the pH vs k(cat)/K(m) rate profile, and is one unit greater than that of the wild-type enzyme, indicating an increase in the local dielectric constant to a value of 21.2 +/- 2.6. A loss of structure of the beta-hairpin from residues 50 to 57, which covers the active site, and is the site of the mutation, is indicated by the disappearance in the F50A mutant of four interstrand NOEs and one turn NOE found in wild-type 4-OT. (1)H-(15)N HSQC spectra of the F50A mutant reveal widespread and large changes in the backbone (15)N and NH chemical shifts including those of Gly residues 48, 51, 53, and 54 causing their loss of dispersion at 23 degrees C and their disappearance at 43 degrees C due to rapid exchange with solvent. These observations confirm that the active site of the F50A mutant is more accessible to the external aqueous environment, causing an increase in the local dielectric constant and in the pK(a) of Pro-1. In addition, the F50A mutation decreased k(cat) 167-fold and increased K(m) 11-fold from those of the wild-type enzyme, suggesting an important role for the hydrophobic environment in catalysis, beyond that of decreasing the pK(a) of Pro-1. The F50I and F50V mutations destabilize the protein and decrease k(cat) by factors of 58 and 1.6, and increase K(m) by 3.3- and 3.8-fold, respectively.
Subject(s)
Isomerases/chemistry , Isomerases/genetics , Mutagenesis, Site-Directed , Phenylalanine/genetics , Alanine/genetics , Amino Acid Sequence , Catalysis , Circular Dichroism , Escherichia coli/enzymology , Escherichia coli/genetics , Hydrogen-Ion Concentration , Isomerases/metabolism , Kinetics , Models, Chemical , Models, Molecular , Molecular Sequence Data , Nitrogen Isotopes , Nuclear Magnetic Resonance, Biomolecular , Phenylalanine/chemistry , Phenylalanine/metabolism , Polymerase Chain Reaction , Proline/metabolism , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Structure-Activity Relationship , Titrimetry , Tyrosine/genetics , Tyrosine/metabolismABSTRACT
Several naturally occurring coumarins, to which humans are routinely exposed in the diet, were previously found to inhibit P450-mediated metabolism of benzo[a]pyrene (B[a]P) and 7,12-dimethylbenz[a]anthracene (DMBA) in vitro, block DNA adduct formation in mouse epidermis and inhibit skin tumor initiation by B[a]P and/or DMBA when applied topically to mice. The present study was designed to investigate the effects of two of these compounds, of the linear furanocoumarin type, when given orally (70 mg/kg per os, four successive daily doses), on P450 and glutathione S-transferase (GST) activities and DNA adduct formation by B[a]P and DMBA in various mouse tissues. Imperatorin and isopimpinellin significantly blocked ethoxyresorufin O-deethylase (EROD) and pentoxyresorufin O:-dealkylase (PROD) activities in epidermis at 1 and 24 h after oral dosing. Imperatorin and isopimpinellin modestly inhibited EROD activities in lung and forestomach at 1 h and significantly inhibited PROD activities in lung and forestomach at 1 h after the final oral dose. Twenty-four hours after the final oral dose of imperatorin or isopimpinellin EROD and PROD activities remained inhibited in epidermis and lung. However, forestomach P450 activity had returned to control levels. Interestingly, imperatorin and isopimpinellin treatment inhibited liver EROD activity at 1 h, had no effect on PROD activity at this time point, but elevated both these enzyme activities at 24 h. Elevated EROD and PROD activities coincided with elevated hepatic P450 content. Imperatorin and isopimpinellin treatment also increased liver cytosolic GST activity at both 1 and 24 h after the final oral dose by 1.6-fold compared with corn oil controls. Oral administration of imperatorin and isopimpinellin also had a protective effect against DNA adduct formation by B[a]P and DMBA. Imperatorin pretreatment decreased formation of DNA adducts by DMBA in forestomach. Pretreatment with isopimpinellin led to reduced DNA adduct levels in liver (B[a]P), lung (B[a]P) and mammary epithelial cells (DMBA). These results suggest that imperatorin and isopimpinellin may have potential chemopreventive effects when administered in the diet.
Subject(s)
9,10-Dimethyl-1,2-benzanthracene/metabolism , Benzo(a)pyrene/metabolism , Cytochrome P-450 Enzyme System/metabolism , DNA Adducts/biosynthesis , Furocoumarins/pharmacology , Glutathione Transferase/metabolism , 9,10-Dimethyl-1,2-benzanthracene/toxicity , Administration, Oral , Animals , Anticarcinogenic Agents/pharmacology , Benzo(a)pyrene/antagonists & inhibitors , Benzo(a)pyrene/toxicity , Carcinogens/metabolism , Carcinogens/toxicity , Cytochrome P-450 CYP1A1/antagonists & inhibitors , Cytochrome P-450 CYP1A1/metabolism , Cytochrome P-450 CYP2B1/antagonists & inhibitors , Cytochrome P-450 CYP2B1/metabolism , Cytochrome P-450 Enzyme Inhibitors , DNA Adducts/antagonists & inhibitors , Female , Liver/drug effects , Liver/enzymology , Lung/drug effects , Lung/enzymology , Mammary Glands, Animal/drug effects , Mammary Glands, Animal/enzymology , Mice , Mice, Inbred SENCAR , Skin/drug effects , Skin/enzymology , Stomach/drug effects , Stomach/enzymologyABSTRACT
DsrA is an 85-nucleotide, untranslated RNA that has multiple regulatory activities at 30 degrees C. These activities include the translational regulation of RpoS and H-NS, global transcriptional regulators in Escherichia coli. Hfq is an E. coli protein necessary for the in vitro and in vivo replication of the RNA phage Qbeta. Hfq also plays a role in the degradation of numerous RNA transcripts. Here we show that an hfq mutant strain is defective for DsrA-mediated regulation of both rpoS and hns. The defect in rpoS expression can be partially overcome by overexpression of DsrA. Hfq does not regulate the transcription of DsrA, and DsrA does not alter the accumulation of Hfq. However, in an hfq mutant, chromosome-expressed DsrA was unstable (half-life of 1 min) and truncated at the 3' end. When expressed from a multicopy plasmid, DsrA was stable in both wild-type and hfq mutant strains, but it had only partial activity in the hfq mutant strain. Purified Hfq binds DsrA in vitro. These results suggest that Hfq acts as a protein cofactor for the regulatory activities of DsrA by either altering the structure of DsrA or forming an active RNA-protein complex.
Subject(s)
Carrier Proteins/genetics , Carrier Proteins/metabolism , Escherichia coli Proteins , Escherichia coli/genetics , Gene Expression Regulation, Bacterial , RNA, Untranslated/genetics , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Chromosomes, Bacterial/genetics , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Escherichia coli/metabolism , Host Factor 1 Protein , Integration Host Factors , Lac Operon/genetics , Mutation , Plasmids/genetics , Protein Biosynthesis , RNA, Small Untranslated , RNA, Untranslated/metabolism , Recombinant Fusion Proteins/metabolism , Sigma Factor/genetics , Sigma Factor/metabolism , Transcription, GeneticABSTRACT
Phenylpyruvate tautomerase (PPT) has been studied periodically since its activity was first described over forty years ago. In the last two years, the mechanism of PPT has been investigated more extensively because of the discovery that PPT is the same protein as the immunoregulatory cytokine known as macrophage migration inhibitory factor (MIF). The mechanism of PPT is likely to involve general base-general acid catalysis. While several lines of evidence implicate Pro-1 as the general base, the identity of the general acid remains unknown. Crystal structures of MIF with the competitive inhibitor (E)-2-fluoro-p-hydroxycinnamate bound in the active site and that of the protein complexed with the enol form of a substrate, (p-hydroxyphenyl)pyruvate, suggest that Tyr-95 is the only candidate in the vicinity that can function as a general acid catalyst. Although Tyr-95 is nearby the bound inhibitor and substrate, it is not within hydrogen bonding distance of either ligand. In this study, Tyr-95 was mutated to phenylalanine, and the kinetic and structural properties of the Y95F mutant were determined. This alteration produces a fully active enzyme, which shows no significant structural changes in the active site. The results indicate that Tyr-95 does not function as the general acid catalyst in the reaction catalyzed by wild-type PPT. The mechanism of PPT was studied further by constructing and characterizing the kinetic properties of two mutants of Pro-1 (P1G and P1A) and one mutant of Asn-97 (N97A). The mutation of Asn-97, a residue implicated in the binding of the phenolic hydroxy group of the keto and enol isomers of (p-hydroxyphenyl)pyruvate and of (E)-2-fluoro-p-hydroxycinnamate affects only the binding affinity of the inhibitor. However, the mutations of Pro-1 have a profound effect on the values of k(cat) and k(cat)/K(m) and clearly show that Pro-1 is a critical residue in the reaction. The results are discussed in terms of a mechanism in which Pro-1 functions as both the general acid and the general base catalyst.
Subject(s)
Intramolecular Oxidoreductases/metabolism , Macrophage Migration-Inhibitory Factors/metabolism , Catalytic Domain/genetics , Cell Migration Inhibition , Intramolecular Oxidoreductases/genetics , Kinetics , Macrophage Migration-Inhibitory Factors/genetics , Models, Chemical , Models, Molecular , Mutation , Phenylalanine/genetics , Proline/genetics , Recombinant Proteins/metabolism , Tyrosine/geneticsSubject(s)
Delivery of Health Care/legislation & jurisprudence , Health Care Reform/legislation & jurisprudence , Health Care Sector/legislation & jurisprudence , Leadership , Delivery of Health Care/organization & administration , Health Care Sector/organization & administration , Humans , New Jersey , Practice Patterns, Physicians' , Societies, MedicalABSTRACT
4-Oxalocrotonate decarboxylase (4-OD) and vinylpyruvate hydratase (VPH) from Pseudomonas putida mt-2 form a complex that converts 2-oxo-3-hexenedioate to 2-oxo-4-hydroxypentanoate in the catechol meta fission pathway. To facilitate mechanistic and structural studies of the complex, the two enzymes have been coexpressed and the complex has been purified to homogeneity. In addition, Glu-106, a potential catalytic residue in VPH, has been changed to glutamine, and the resulting E106QVPH mutant has been coexpressed with 4-OD and purified to homogeneity. The 4-OD/E106QVPH complex retains full decarboxylase activity, with comparable kinetic parameters to those observed for 4-OD in the wild-type complex, but is devoid of any detectable hydratase activity. Decarboxylation of (5S)-2-oxo-3-[5-D]hexenedioate by either the 4-OD/VPH complex or the mutant complex generates 2-hydroxy-2,4E-[5-D]pentadienoate in D(2)O. Ketonization of 2-hydroxy-2,4-pentadienoate by the wild-type complex is highly stereoselective and results in the formation of 2-oxo-(3S)-[3-D]-4-pentenoate, while the mutant complex generates a racemic mixture. These results indicate that 2-hydroxy-2, 4-pentadienoate is the product of 4-OD and that 2-oxo-4-pentenoate results from a VPH-catalyzed process. On this basis, the previously proposed hypothesis for the conversion of 2-oxo-3-hexenedioate to 2-oxo-4-hydroxypentanoate has been revised [Lian, H., and Whitman, C. P. (1994) J. Am. Chem. Soc. 116, 10403-10411]. Finally, the observed (13)C kinetic isotope effect on the decarboxylation of 2-oxo-3-hexenedioate by the 4-OD/VPH complex suggests that the decarboxylation step is nearly rate-limiting. Because the value is not sensitive to either magnesium or manganese, it is likely that the transition state for carbon-carbon bond cleavage is late and that the metal positions the substrate and polarizes the carbonyl group, analogous to its role in oxalacetate decarboxylase.
Subject(s)
Carboxy-Lyases/biosynthesis , Carboxy-Lyases/chemistry , Carbon Isotopes , Carboxy-Lyases/genetics , Deuterium , Enzyme Activation/genetics , Escherichia coli/genetics , Genetic Vectors/chemical synthesis , Glutamic Acid/chemistry , Glutamic Acid/genetics , Glutamine/chemistry , Glutamine/genetics , Hydro-Lyases/genetics , Kinetics , Mutagenesis, Site-Directed , Nuclear Magnetic Resonance, Biomolecular , Protons , Pseudomonas putida/enzymology , Pseudomonas putida/genetics , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemical synthesis , Recombinant Proteins/isolation & purification , StereoisomerismABSTRACT
Macrophage migration inhibitory factor (MIF) is a cytokine that was first described as an inhibitor of the random migration of monocytes and macrophages and has since been proposed to have a number of immune and catalytic functions. One of the functions assigned to MIF is that of a tautomerase that interconverts the enol and keto forms of phenylpyruvate and (p-hydroxyphenyl)pyruvate and converts D-dopachrome, a stereoisomer of naturally occurring L-dopachrome, to 5,6-dihydroxyindole-2-carboxylic acid. The physiological significance of the MIF enzymatic activity is unclear. The three-dimensional structure of MIF is strikingly similar to that of two microbial enzymes (4-oxalocrotonate tautomerase and 5-carboxymethyl-2-hydroxymuconate isomerase) that otherwise share little sequence identity with MIF. MIF and these two enzymes have an invariant N-terminal proline that serves as a catalytic base. Here we report a new biological function for MIF, as an inhibitor of monocyte chemoattractant protein 1- (MCP-1-) induced chemotaxis of human peripheral blood monocytes. We find that MIF inhibition of chemotaxis does not occur at the level of the CC chemokine receptor for MCP-1, CCR2, since MIF does not alter the binding of (125)I-MCP-1 to monocytes. The role of MIF enzymatic activity in inhibition of monocyte chemotaxis and random migration was studied with two MIF mutants in which the N-terminal proline was replaced with either a serine or a phenylalanine. Both mutants remain capable of inhibiting monocyte chemotaxis and random migration despite significantly reduced or no phenylpyruvate tautomerase activity. These data suggest that this enzymatic activity of MIF does not play a role in its migration inhibiting properties.
Subject(s)
Cell Movement/drug effects , Chemotaxis, Leukocyte/drug effects , Macrophage Migration-Inhibitory Factors/pharmacology , Receptors, Chemokine , Animals , Base Sequence , Chemokine CCL2/metabolism , Circular Dichroism , DNA Primers , Humans , In Vitro Techniques , Kinetics , Mass Spectrometry , Mice , Receptors, CCR2 , Receptors, Cytokine/metabolism , Recombinant Proteins/pharmacologyABSTRACT
Three arginine residues (Arg-11, Arg-39, Arg-61) are found at the active site of 4-oxalocrotonate tautomerase in the X-ray structure of the affinity-labeled enzyme [Taylor, A. B., Czerwinski, R. M., Johnson, R. M., Jr., Whitman, C. P., and Hackert, M. L. (1998) Biochemistry 37, 14692-14700]. The catalytic roles of these arginines were examined by mutagenesis, kinetic, and heteronuclear NMR studies. With a 1,6-dicarboxylate substrate (2-hydroxymuconate), the R61A mutation showed no kinetic effects, while the R11A mutation decreased k(cat) 88-fold and increased K(m) 8.6-fold, suggesting both binding and catalytic roles for Arg-11. With a 1-monocarboxylate substrate (2-hydroxy-2,4-pentadienoate), no kinetic effects of the R11A mutation were found, indicating that Arg-11 interacts with the 6-carboxylate of the substrate. The stereoselectivity of the R11A-catalyzed protonation at C-5 of the dicarboxylate substrate decreased, while the stereoselectivity of protonation at C-3 of the monocarboxylate substrate increased in comparison with wild-type 4-OT, indicating the importance of Arg-11 in properly orienting the dicarboxylate substrate by interacting with the charged 6-carboxylate group. With 2-hydroxymuconate, the R39A and R39Q mutations decreased k(cat) by 125- and 389-fold and increased K(m) by 1.5- and 2.6-fold, respectively, suggesting a largely catalytic role for Arg-39. The activity of the R11A/R39A double mutant was at least 10(4)-fold lower than that of the wild-type enzyme, indicating approximate additivity of the effects of the two arginine mutants on k(cat). For both R11A and R39Q, 2D (1)H-(15)N HSQC and 3D (1)H-(15)N NOESY-HSQC spectra showed chemical shift changes mainly near the mutated residues, indicating otherwise intact protein structures. The changes in the R39Q mutant were mainly in the beta-hairpin from residues 50 to 57 which covers the active site. HSQC titration of R11A with the substrate analogue cis, cis-muconate yielded a K(d) of 22 mM, 37-fold greater than the K(d) found with wild-type 4-OT (0.6 mM). With the R39Q mutant, cis, cis-muconate showed negative cooperativity in active site binding with two K(d) values, 3.5 and 29 mM. This observation together with the low K(m) of 2-hydroxymuconate (0.47 mM) suggests that only the tight binding sites function catalytically in the R39Q mutant. The (15)Nepsilon resonances of all six Arg residues of 4-OT were assigned, and the assignments of Arg-11, -39, and -61 were confirmed by mutagenesis. The binding of cis,cis-muconate to wild-type 4-OT upshifts Arg-11 Nepsilon (by 0.05 ppm) and downshifts Arg-39 Nepsilon (by 1.19 ppm), indicating differing electronic delocalizations in the guanidinium groups. A mechanism is proposed in which Arg-11 interacts with the 6-carboxylate of the substrate to facilitate both substrate binding and catalysis and Arg-39 interacts with the 1-carboxylate and the 2-keto group of the substrate to promote carbonyl polarization and catalysis, while Pro-1 transfers protons from C-3 to C-5. This mechanism, together with the effects of mutations of catalytic residues on k(cat), provides a quantitative explanation of the 10(7)-fold catalytic power of 4-OT. Despite its presence in the active site in the crystal structure of the affinity-labeled enzyme, Arg-61 does not play a significant role in either substrate binding or catalysis.
Subject(s)
Arginine/genetics , Isomerases/chemistry , Isomerases/genetics , Mutagenesis, Site-Directed , Alanine/genetics , Binding Sites/genetics , Catalysis , Glutamine/genetics , Isomerases/biosynthesis , Isomerases/metabolism , Kinetics , Models, Molecular , Nitrogen Isotopes , Nuclear Magnetic Resonance, Biomolecular , Pseudomonas putida/enzymology , Pseudomonas putida/genetics , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemical synthesis , Recombinant Proteins/chemistry , Sorbic Acid/analogs & derivatives , Sorbic Acid/metabolism , Stereoisomerism , TitrimetryABSTRACT
The unusually low pK(a) value of the general base catalyst Pro-1 (pK(a) = 6.4) in 4-oxalocrotonate tautomerase (4-OT) has been ascribed to both a low dielectric constant at the active site and the proximity of the cationic residues Arg-11 and Arg-39 [Stivers, J. T., Abeygunawardana, C., Mildvan, A. S., Hajipour, G., and Whitman, C. P. (1996) Biochemistry 35, 814-823]. In addition, the pH-rate profiles in that study showed an unidentified protonated group essential for catalysis with a pK(a) of 9.0. To address these issues, the pK(a) values of the active site Pro-1 and lower limit pK(a) values of arginine residues were determined by direct (15)N NMR pH titrations. The pK(a) values of Pro-1 and of the essential acid group were determined independently from pH-rate profiles of the kinetic parameters of 4-OT in arginine mutants of 4-OT and compared with those of wild type. The chemical shifts of all of the Arg Nepsilon resonances in wild-type 4-OT and in the R11A and R39Q mutants were found to be independent of pH over the range 4.9-9.7, indicating that no arginine is responsible for the kinetically determined pK(a) of 9.0 for an acidic group in free 4-OT. With the R11A mutant, where k(cat)/K(m) was reduced by a factor of 10(2.9), the pK(a) of Pro-1 was not significantly altered from that of the wild-type enzyme (pK(a) = 6.4 +/- 0.2) as revealed by both direct (15)N NMR titration (pK(a) = 6.3 +/- 0.1) and the pH dependence of k(cat)/K(m) (pK(a) = 6.4 +/- 0.2). The pH-rate profiles of both k(cat)/K(m) and k(cat) for the reaction of the R11A mutant with the dicarboxylate substrate, 2-hydroxymuconate, showed humps, i.e., sharply defined maxima followed by nonzero plateaus. The humps disappeared in the reaction with the monocarboxylate substrate, 2-hydroxy-2,4-pentadienoate, indicating that, unlike the wild-type enzyme which reacts only with the dianionic form of the dicarboxylic substrate, the R11A mutant reacts with both the 6-COOH and 6-COO(-) forms, with the 6-COOH form being 12-fold more active. This reversal in the preferred ionization state of the 6-carboxyl group of the substrate that occurs upon mutation of Arg-11 to Ala provides strong evidence that Arg-11 interacts with the 6-carboxylate of the substrate. In the R39Q mutant, where k(cat)/K(m) was reduced by a factor of 10(3), the kinetically determined pK(a) value for Pro-1 was 4.6 +/- 0.2, while the ionization of Pro-1 showed negative cooperativity with an apparent pK(a) of 7.1 +/- 0.1 determined by 1D (15)N NMR. From the Hill coefficient of 0.54, it can be shown that the apparent pK(a) value of 7.1 could result most simply from the averaging of two limiting pK(a) values of 4.6 and 8.2. Mutation of Arg-39, by altering the structure of the beta-hairpin which covers the active site, could result in an increase in the solvent exposure of Pro-1, raising its upper limit pK(a) value to 8.2. In the R39A mutant, the kinetically determined pK(a) of Pro-1 was also low, 5.0 +/- 0.2, indicating that in both the R39Q and R39A mutants, only the sites with low pK(a) values were kinetically operative. With the fully active R61A mutant, the kinetically determined pK(a) of Pro-1 (pK(a) = 6.5 +/- 0.2) agreed with that of wild-type 4-OT. It is concluded that the unusually low pK(a) of Pro-1 shows little contribution from electrostatic effects of the nearby cationic Arg-11, Arg-39, and Arg-61 residues but results primarily from a site of low local dielectric constant.
Subject(s)
Arginine/genetics , Arginine/metabolism , Isomerases/genetics , Isomerases/metabolism , Mutagenesis, Site-Directed , Alanine/genetics , Binding Sites/genetics , Catalysis , Glutamine/metabolism , Hydrogen-Ion Concentration , Kinetics , Nitrogen Isotopes , Nuclear Magnetic Resonance, Biomolecular , Proline/metabolism , Pseudomonas putida/enzymology , Pseudomonas putida/genetics , Substrate Specificity , TitrimetryABSTRACT
Macrophage migration inhibitory factor (MIF) exhibits dual activities. It acts as an immunoregulatory protein as well as a phenylpyruvate tautomerase. To understand better the relationship between these two activities and to elucidate the structural basis for the enzymatic activity, a crystal structure of a complex between murine MIF and (E)-2-fluoro-p-hydroxycinnamate, a competitive inhibitor of the tautomerase activity, has been determined to 1.8 A resolution. The structure is nearly superimposable on that of the free protein indicating that the presence of the inhibitor does not result in any major structural changes. The inhibitor also confirms the location of the active site in a hydrophobic cavity containing the amino-terminal proline. Within this cavity, the inhibitor interacts with residues from adjacent subunits. At the back of the cavity, the side-chain carbonyl oxygen of Asn-97' interacts with the phenolic hydroxyl group of the inhibitor while at the mouth of the cavity the ammonium group of Lys-32 interacts with a carboxylate oxygen. The other carboxylate oxygen of the inhibitor interacts with Pro-1. The hydroxyl group of Tyr-95' interacts weakly with the fluoro group on the inhibitor. The hydrophobic side chains of five active-site residues (Met-2, Ile-64, Met-101, Val-106, and Phe-113) and the phenyl moiety of Tyr-95' are responsible for the binding of the phenyl group. Further insight into the enzymatic activity of MIF was obtained by carrying out kinetic studies using the enol isomers of phenylpyruvate and (p-hydroxyphenyl)pyruvate. The results demonstrate that MIF processes the enol isomers more efficiently than the keto isomers primarily because of a decrease in Km. On the basis of these results, a mechanism is proposed for the MIF-catalyzed tautomerization reaction.
Subject(s)
Cinnamates/chemistry , Coumaric Acids/chemistry , Enzyme Inhibitors/chemistry , Intramolecular Oxidoreductases/antagonists & inhibitors , Macrophage Migration-Inhibitory Factors/chemistry , Macrophage Migration-Inhibitory Factors/metabolism , Animals , Binding, Competitive , Catalysis , Cinnamates/metabolism , Coumaric Acids/metabolism , Crystallization , Crystallography, X-Ray , Enzyme Inhibitors/metabolism , Fatty Acids, Unsaturated/chemistry , Intramolecular Oxidoreductases/chemistry , Intramolecular Oxidoreductases/metabolism , Isomerases/antagonists & inhibitors , Kinetics , Macromolecular Substances , Macrophage Migration-Inhibitory Factors/antagonists & inhibitors , Mice , Models, Molecular , Protein Binding , Sequence Homology, Amino AcidABSTRACT
2-oxo-3-pentynoate has been characterized as an active-site-directed inhibitor of selected flavoprotein oxidases. Tryptophan 2-monooxygenase is irreversibly inactivated in an active-site-directed fashion. The addition of FAD affords no protection from inactivation, whereas the competitive inhibitor indole-3-acetamide fully protects the enzyme from inactivation. The inactivation follows first-order kinetics for at least five half-lives. The rate of inactivation shows saturation kinetics, consistent with the formation of a reversible complex between the alkylating agent and the enzyme before inactivation occurs. Values of 0.017 +/- 0.0005 min-1 and 44 +/- 7 microM were determined for the limiting rate of inactivation and the apparent dissociation constant for 2-oxo-3-pentynoate, respectively. Tryptic maps of tryptophan 2-monooxygenase treated with 2-oxo-3-pentynoate show that two peptides are alkylated in the absence of indole-3-acetamide but not in its presence. The two peptides were identified by mass spectrometry as residues 333-349 and 503-536. Based upon sequence analysis, cysteine 511 and either cysteine 339 or histidine 338 are the likely sites of modification. In contrast, incubation of D-amino acid oxidase or nitroalkane oxidase with 2-oxo-3-pentynoate results in a loss of 55% or 100%, respectively, of the initial activity. In neither case does a competitive inhibitor affect the rate of inactivation, suggesting that the effect is not due to modification of active-site residues.
Subject(s)
Dioxygenases , Enzyme Inhibitors/chemistry , Fatty Acids, Unsaturated/chemistry , Mixed Function Oxygenases/antagonists & inhibitors , Peptide Fragments/chemistry , Alkylation , Amino Acid Sequence , Binding Sites , D-Amino-Acid Oxidase/antagonists & inhibitors , Enzyme Activation , Molecular Sequence Data , Oxygenases/antagonists & inhibitors , Pseudomonas/enzymology , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
This article reports on a new instrument, the Geriatric Institutional Assessment Profile (GIAP), developed to assess (1) hospital workers' knowledge, attitudes, and perceptions regarding care of geriatric patients, and (2) the perceived adequacy of an institutional environment to serve geriatric patients' needs. Findings are reported from 303 questionnaires completed by health care employees from a 658-bed academic medical center. Internal consistency estimates were consistently high for the various components of the GIAP. Factor analysis was performed to examine underlying dimensions of knowledge and institutional environment. The GIAP has the potential to narrow the gap between actual and best practice in geriatric care by identifying staff information needs and concerns, as well as institutional barriers and facilitators to providing quality geriatric hospital care.
Subject(s)
Benchmarking/standards , Geriatric Nursing/standards , Health Services for the Aged/standards , Adult , Aged , Female , Humans , Male , Middle Aged , Reproducibility of Results , Surveys and Questionnaires/standards , United StatesABSTRACT
Macrophage migration inhibitory factor (MIF), an immunoregulatory protein, exhibits a phenylpyruvate tautomerase (PPT) activity. The catalytic mechanism of this activity has recently attracted attention in an effort to determine whether there is a relationship between the PPT activity and the role of MIF in various immune and inflammatory processes. One of the active site residues is lysine-32, which is postulated to play two roles: it assists in substrate binding through an interaction with a carboxylate oxygen at C-1 of phenylpyruvate, and it may be partially responsible for lowering the pK(a) of the catalytic base, Pro-1. The role of Lys-32 has been investigated by changing it to an alanine and an arginine and determining the kinetic parameters, the stereoselectivity, the competitive inhibition, and the pH dependence of the resulting K32A- and K32R-catalyzed reactions. For the K32R mutant, these properties are mostly comparable to those determined for the wild type with two exceptions. There is a modest decrease in the stereoselectivity of the reaction and in the binding affinity of the competitive inhibitor, (E)-2-fluoro-p-hydroxycinnamate. These differences are likely due to the increased steric bulk of arginine. For the K32A mutant, there are 11- and 12-fold decreases in k(cat) and k(cat)/K(m), respectively, using phenylenolpyruvate. Part of the decrease in activity can be attributed to the observed increase of 1. 3 units in the pK(a) of Pro-1. It was also found that the loss of the electrostatic interaction did not significantly affect the stereoselectivity of the K32A-catalyzed reaction, although it did result in a decrease in the binding affinity of the competitive inhibitor. The combination of these results indicates that the primary function of Lys-32 in the PPT activity of MIF is to lower the pK(a) of Pro-1. The interactions responsible for the stereoselectivity of the PPT activity were further delineated by examining the wild type- and K32A-catalyzed reactions with an alternate substrate, 2-hydroxy-2,4-pentadienoate, in which the phenyl group of phenylenolpyruvate is replaced with a double bond. The effect of this substitution is moderate as evidenced by the observation that the ketonization of 2-hydroxy-2,4-pentadienoate by the wild type protein is more stereoselective than the K32R-catalyzed ketonization of phenylenolpyruvate but not as stereoselective as the K32A-catalyzed ketonization of phenylenolpyruvate. However, the low degree of stereoselectivity observed for the K32A-catalyzed reaction indicates that an electrostatic interaction between the protein and 2-hydroxy-2, 4-pentadienoate is now crucial.
Subject(s)
Intramolecular Oxidoreductases/metabolism , Macrophage Migration-Inhibitory Factors/metabolism , Animals , Binding Sites , Binding, Competitive , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Hydrogen-Ion Concentration , Intramolecular Oxidoreductases/antagonists & inhibitors , Intramolecular Oxidoreductases/chemistry , Kinetics , Lysine/chemistry , Macrophage Migration-Inhibitory Factors/biosynthesis , Macrophage Migration-Inhibitory Factors/chemistry , Magnetic Resonance Spectroscopy , Mice , Molecular Conformation , Mutation , StereoisomerismABSTRACT
The crystal structure of 4-oxalocrotonate tautomerase (4-OT) inactivated by the active site-directed irreversible inhibitor 2-oxo-3-pentynoate (2-OP) has been determined to 2.4 A resolution. The structure of the enzyme covalently modified at Pro-1 by the resulting 2-oxo-3-pentenoate adduct is nearly superimposable on that of the free enzyme and confirms that the active site is located in a hydrophobic region surrounding Pro-1. Both structures can be described as a trimer of dimers where each dimer consists of a four-stranded beta-sheet with two antiparallel alpha-helices on one side. Examination of the structure also reveals noncovalent interactions between the adduct and two residues in the active site. The epsilon and eta nitrogens of the guanidinium side chain of Arg-39" from a neighboring dimer interact respectively with the C-2 carbonyl oxygen and one C-1 carboxylate oxygen of the adduct while the side chain of Arg-61' from the same dimer as the modified Pro-1 interacts with the C-1 carboxylate group in a bidentate fashion. An additional interaction to the 2-oxo group of the adduct is provided by one of the two ordered water molecules within the active site region. These interactions coupled with the observation that 2-oxo-3-butynoate is a more potent irreversible inhibitor of 4-oxalocrotonate tautomerase than is 2-OP suggest that Arg-39" and the ordered water molecule polarize the carbonyl group of 2-OP which facilitates a Michael reaction between Pro-1 and the acetylene compound. On the basis of the crystal structure, a mechanism for the enzyme-catalyzed reaction is proposed.
Subject(s)
Enzyme Inhibitors/pharmacology , Fatty Acids, Unsaturated/pharmacology , Isomerases/antagonists & inhibitors , Isomerases/chemistry , Binding Sites , Catalysis , Crystallization , Crystallography, X-Ray , Enzyme Activation/drug effects , Kinetics , Models, Molecular , Pseudomonas putida/enzymology , Structure-Activity Relationship , Substrate SpecificityABSTRACT
The cytokine macrophage migration inhibitory factor (MIF) mediates several immune and inflammatory processes through unknown or poorly understood mechanisms. The protein shares structural homology with two bacterial isomerases, 4-oxalocrotonate tautomerase (4-OT) and 5-(carboxymethyl)-2-hydroxymuconate isomerase (CHMI), and catalyzes the enolization of phenylpyruvate and the ketonization of (p-hydroxyphenyl)pyruvate. The amino-terminal proline has been identified as the catalytic base in both the 4-OT- and CHMI-catalyzed reactions. MIF also has an amino-terminal proline that has been implicated as a catalytic group in the MIF-catalyzed reaction. To delineate further the role of Pro-1 in the MIF-catalyzed reaction, affinity labeling studies were performed with 3-bromopyruvate (3-BP). The results of this study show that 3-BP acts as an active-site-directed irreversible inhibitor of the enzymatic activity and modifies one site per monomeric subunit. The inhibitor, as its lactyl derivative, is covalently attached to an 11 residue amino-terminal fragment, Pro-1 to Arg-11. The only reasonable site for alkylation within this peptide fragment is the amino-terminal proline. Because the pKa measured for the pH dependence of kinact/KI (5.7 +/- 0.2) and that measured for the pH dependence of the kcat/Km for the enolization of phenylpyruvate (6.0 +/- 0.1) are comparable and in reasonable agreement with the previously measured pKa of Pro-1 (5.6 +/- 0.1) obtained by its direct titration [Swope, M., Sun H.-W., Blake, P., and Lolis, E. (1998) EMBO J. (in press)], it is concluded that Pro-1 acts as the general base catalyst in the MIF-catalyzed reaction. The structural and mechanistic parallels place 4-OT, CHMI, and MIF in a superfamily of enzymes related by their ability to catalyze the keto-enol tautomerization of a pyruvyl moiety.
Subject(s)
Carbon-Carbon Double Bond Isomerases/metabolism , Isomerases/metabolism , Macrophage Migration-Inhibitory Factors/metabolism , Peptide Fragments/metabolism , Proline/metabolism , Animals , Carbon-Carbon Double Bond Isomerases/antagonists & inhibitors , Carbon-Carbon Double Bond Isomerases/chemistry , Catalysis , Enzyme Activation/drug effects , Hydrogen-Ion Concentration , Isomerases/antagonists & inhibitors , Isomerases/chemistry , Kinetics , Macrophage Migration-Inhibitory Factors/chemistry , Mice , Peptide Fragments/chemistry , Peptide Fragments/isolation & purification , Proline/chemistry , Pyruvates/pharmacology , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Substrate SpecificityABSTRACT
pp120, a substrate of the insulin receptor tyrosine kinase, does not undergo ligand-stimulated phosphorylation by the insulin-like growth factor-1 (IGF-1) receptor. However, replacement of the C-terminal domain of the IGF-1 receptor beta-subunit with the corresponding segment of the insulin receptor restored pp120 phosphorylation by the chimeric receptor. Since pp120 stimulates receptor-mediated insulin endocytosis when it is phosphorylated, we examined whether pp120 regulates IGF-1 receptor endocytosis in transfected NIH 3T3 cells. pp120 failed to alter IGF-1 receptor endocytosis via either wild-type or chimeric IGF-1 receptors. Thus, the effect of pp120 on hormone endocytosis is specific to insulin, and the C-terminal domain of the beta-subunit of the insulin receptor does not regulate the effect of pp120 on insulin endocytosis. Mutation of Tyr960 in the juxtamembrane domain of the insulin receptor abolished the effect of pp120 to stimulate receptor endocytosis, without affecting pp120 phosphorylation by the insulin receptor. These findings suggest that pp120 interacts with two separate domains of the insulin receptor as follows: a C-terminal domain required for pp120 phosphorylation and a juxtamembrane domain required for internalization. We propose that the interaction of pp120 with the juxtamembrane domain is indirect and requires one or more substrates that bind to Tyr960 in the insulin receptor.