Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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A series of silica gels and mesoporous molecular sieves differing in both the range of particle size and mean pore size were derivatized with the p-[R,S-alpha-[1-(9H-fluoren-9-yl)-methoxyformamido]-2,4-di methoxybenzyl]- phenoxyacetic acid linker and their loading capacities were measured. Loading capacities ranging between 0.4-0.6 mmol Fmoc/g were achieved. Several of these silica based materials were derivatized with the hydroxymethyl benzoic acid linker and used as supports for the solid phase Claisen rearrangement of a support bound phenyl allyl ether. Both the silica gel and mesoporous supports were heated at 225 degrees C for 3 h to effect the Claisen rearrangement. The results showed that, compared to the same reaction run homogeneously, the silica gel support achieved similar total product yields and ratios for two Claisen products. The mesoporous supports were found to selectively produce one of the Claisen products over the other. Analysis shows that the molecules bound to the mesoporous support are physically further separated from each other as compared to those bound to the silica gel support. A mechanism is presented which accounts for the selectivity of the mesoporous support in forming one Claisen product over the other. The Claisen product was further derivatized to the resulting phenyl ethyl either through a solid phase Mitsunobu reaction on the mesoporous support.
Mol Divers
PMID:Use of silica gels and mesoporous molecular sieves as supports for the solid-phase Claisen rearrangement. 968 Jun 47

A mutation in the dimer interface of Escherichia coli glycinamide ribonucleotide transformylase (GarTfase) disrupts the observed pH-dependent association of the wild-type enzyme, but has no observable effect on the enzyme activity. Here, we assess whether a pH effect on the enzyme's conformation is sufficient by itself to explain the pH-dependence of the GarTfase reaction. A pH-dependent conformational change is observed between two high-resolution crystal structures of the Glu70Ala mutant GarTfase at pH 3.5 (1.8 A) and 7.5 (1.9 A). Residues 110 to 131 in GarTfase undergo a transformation from a disordered loop at pH 3.5, where the enzyme is inactive, to an ordered loop-helix structure at pH 7.5, where the enzyme is active. The ordering of this flexible loop-helix has a direct effect on catalytic residues in the active site, binding of the folate cofactor and shielding of the active site from solvent. A main-chain carbonyl oxygen atom from Tyr115 in the ordered loop forms a hydrogen bond with His108, and thereby provides electronic and structural stabilization of this key active site residue. Kinetic data indicate that the pKa of His108 is in fact raised to 9. 2. The loop movement can be correlated with elevation of the His pKa, but with further stabilization, probably from Asp144, after the binding of folate cofactor. Leu118, also in the loop, becomes positioned near the p-amino benzoic acid binding site, providing additional hydrophobic interactions with the cofactor 10-formyl tetrahydrofolate. Thus, the pH-dependence of the enzyme activity appears to arise from local active site rearrangements and not from differences due to monomer-dimer association.
J Mol Biol 1998 Aug 21
PMID:A pH-dependent stabilization of an active site loop observed from low and high pH crystal structures of mutant monomeric glycinamide ribonucleotide transformylase at 1.8 to 1.9 A. 969 64

The therapeutic potential of drugs that block the induction of cyclooxygenase-2 has been emphasized. When two 4-trifluoromethyl salicylate derivatives [2-acetoxy-4-trifluoromethyl-benzoic acid (triflusal) and its deacetylated metabolite 2-hydroxy-4-trifluoromethylbenzoic acid (HTB)] were compared with aspirin and sodium salicylate as cyclooxygenase-2 (COX-2) inhibitors, we observed that in bacterial lipopolysaccharide-activated human blood, triflusal, aspirin, and HTB, but not sodium salicylate, inhibited COX-2-mediated prostaglandin E2 (PGE2) production (IC50 = 0.16, 0.18, 0.39, and >10 mM, respectively). However, only triflusal and aspirin inhibited purified COX-2 enzyme. To test this apparent discrepancy, we realized that HTB and triflusal (but neither aspirin nor salicylate) produced a concentration-dependent inhibition of COX-2 protein expression in peripheral human mononuclear cells. This observation was further confirmed in a rat air pouch model in vivo, in which both aspirin and triflusal inhibited PGE2 production (ID50 = 18.9 and 11.4 mg/kg p.o., respectively) but only triflusal-treated animals showed a decrease in COX-2 expression. This different behavior may be, at least in part, due to the ability of HTB and triflusal to block the activation of the transcription factor nuclear factor-kappaB to a higher extent than aspirin and sodium salicylate. Thus, in addition to inhibiting the COX-2 activity at therapeutic concentrations, triflusal is able to block through its metabolite HTB the expression of new enzyme, and hence the resumption of PGE2 synthesis. Triflusal and HTB may exert beneficial effects in processes in which de novo COX-2 expression is involved and, in a broader sense, in pathological situations in which genes under nuclear factor-kappaB control are up-regulated.
Mol Pharmacol 1999 Apr
PMID:Inhibition of cyclooxygenase-2 expression by 4-trifluoromethyl derivatives of salicylate, triflusal, and its deacetylated metabolite, 2-hydroxy-4-trifluoromethylbenzoic acid. 1010 Oct 34

Human glutathione transferase A1-1 (GST A1-1) is a detoxifying enzyme catalyzing the conjugation of glutathione with a variety of hydrophobic, electrophilic substrates. When the role of the hydrophobic substrate-binding site residue Met208 was investigated by random mutagenesis, introduction of charged amino acid residues had the greatest deleterious effect on enzyme activity. However, in the lysine mutant some of the lost activity could be regained by the addition of a benzoic acid derivative to the reaction mixture. The activating molecule has now been optimized such that all activity is recovered. The most potent activator, 4-propylbenzoic acid, has been used in studies of the mechanism behind the activation. A heterodimeric species of GST A1-1, containing only one activatable subunit, has been constructed. The heterodimer shows a strictly additive activation curve when compared to its parental forms, indicating that the activation is not due to co-operativity between the subunits. Furthermore, a novel electrophilic substrate, 4-chloro-3,5-dinitrobenzoic acid, with a carboxylate group expected to interact with residue 208 gives a higher kcat value with the lysine mutant than with wild-type GST A1-1. All results obtained in the here support the view that the positive charge introduced into the lysine mutant adversely affects the structure of the C-terminal helix of this enzyme, preventing it from adopting the conformation needed for full activity. The negatively charged carboxylate group of the activator probably neutralizes the positive charge of the side-chain amino group and thereby restores the substrate-binding site to a form that is favorable for the catalytic function.
J Mol Biol 1999 May 14
PMID:Benzoic acid derivatives induce recovery of catalytic activity in the partially inactive Met208Lys mutant of human glutathione transferase A1-1. 1032 79

Hypoglycemic sulfonylureas (e.g., glibenclamide, glipizide, and tolbutamide) exert their stimulatory effect on excitatory cells by closure of ATP-sensitive potassium (KATP) channels. These channels are heteromultimers composed with a 4:4 stoichiometry of an inwardly rectifying K+ channel (KIR) subunit 6.x plus a sulfonylurea receptor (SUR). SUR1/KIR6.2 reconstitutes the neuronal/pancreatic beta-cell channel, whereas SUR2A/KIR6.2 and SUR2B/KIR6.1 (or KIR6.2) are proposed to reconstitute the cardiac and the vascular smooth muscle-type KATP channels, respectively. SUR2A and SUR2B are splice variants of a single gene differing only in their C-terminal 42 amino acids. Affinities of sulfonylureas for rat SUR2A, rat or human SUR2B, and a SUR2 chimera containing the C-terminal 42 amino acids of SUR1 did not differ significantly, implying that the C terminus does not form part of the binding pocket. Consistent with these findings, reconstituted SUR2A/KIR6.2 and SUR2B/KIR6.2 channels revealed similar sensitivities for glibenclamide and tolbutamide. Dissociation constants of sulfonylureas for SUR2A and SUR2B were 10- to 400-fold higher than for SUR1, however, amazingly the benzoic acid derivative meglitinide did not show lower affinity for SUR2 isoforms. Potencies of glibenclamide, glipizide, tolbutamide, and meglitinide to inhibit activity of SUR1/KIR6.2 and SUR2B/KIR6.2 channels were 3- to 6-fold higher than binding affinities of these drugs with concentration-inhibition relations being significantly steeper (Hill coefficients 1.23-1.32) than binding curves (Hill coefficients 0.93-1.06). The data establish that the C terminus of SURs does not affect sulfonylurea affinity and sensitivity. We conclude that occupation of one of the four SUR sites per channel complex is sufficient to induce KATP channel closure.
Mol Pharmacol 1999 Jun
PMID:Stoichiometry of sulfonylurea-induced ATP-sensitive potassium channel closure. 1034 49

Tetracaine (N,N-dimethylaminoethyl-4-butylaminobenzoate) and related N,N-dialkylaminoethyl substituted benzoic acid esters have been used to characterize the high-affinity binding site for aromatic amine noncompetitive antagonists in the Torpedo nicotinic acetylcholine receptor (nAChR). [(3)H]Tetracaine binds at equilibrium to a single site with a K(eq) value of 0.5 microM in the absence of agonist or presence of alpha-bungarotoxin and with a K(eq) value of 30 microM in the presence of agonist (i.e., for nAChR in the desensitized state). Preferential binding to nAChR in the absence of agonist is also seen for N,N-DEAE and N,N-diethylaminopropyl esters, both binding with 10-fold higher affinity in the absence of agonist than in the presence, and for the 4-ethoxybenzoic acid ester of N, N-diethylaminoethanol, but not for the 4-amino benzoate ester (procaine). Irradiation at 302 nm of nAChR-rich membranes equilibrated with [(3)H]tetracaine resulted in covalent incorporation with similar efficiency into nAChR alpha, beta, gamma, and delta subunits. The pharmacological specificity of nAChR subunit photolabeling as well as its dependence on [(3)H]tetracaine concentration establish that the observed photolabeling is at the high-affinity [(3)H]tetracaine-binding site. Within alpha subunit, >/=95% of specific photolabeling was contained within a 20-kilodalton proteolytic fragment beginning at Ser(173) that contains the M1 to M3 hydrophobic segments. With all four subunits contributing to [(3)H]tetracaine site, the site in the closed channel state of the nAChR is most likely within the central ion channel domain.
Mol Pharmacol 1999 Aug
PMID:Photoaffinity labeling the torpedo nicotinic acetylcholine receptor with [(3)H]tetracaine, a nondesensitizing noncompetitive antagonist. 1041 47

The active site of type A or B influenza virus neuraminidase is composed of 11 conserved residues that directly interact with the substrate, sialic acid. An aromatic benzene ring has been used to replace the pyranose of sialic acid in our design of novel neuraminidase inhibitors. A bis(hydroxymethyl)pyrrolidinone ring was constructed in place of the N-acetyl group on the sialic acid. The hydroxymethyl groups replace two active site water molecules, which resulted in the high affinity of the nanomolar inhibitors. However, these inhibitors have greater potency for type A influenza virus than for type B influenza virus. To resolve the differences, we determined the X-ray crystal structure of three benzoic acid substituted inhibitors bound to the active site of B/Lee/40 neuraminidase. The investigation of a hydrophobic aliphatic group and a hydrophilic guanidino group on the aromatic inhibitors shows changes in the interaction with the active site residue Glu275. The results provide an explanation for the difference in efficacy of these inhibitors against types A and B viruses, even though the 11 active site residues of the neuraminidase are conserved.
J Mol Biol 1999 Nov 12
PMID:Novel aromatic inhibitors of influenza virus neuraminidase make selective interactions with conserved residues and water molecules in the active site. 1054 89

In situ infrared spectroscopy has been used to investigate the adsorption of a range of simple aromatic carboxylic acids from aqueous solution to metal oxides. Thin films of TiO2, ZrO2, Al2O3 and Ta2O5 were prepared by evaporation of aqueous sols on single reflection ZnSe prisms. Benzoic acid adsorbed very strongly to ZrO2, in a bridging bidentate fashion, but showed only weak adsorption to TiO2 and Ta2O5. Substituted aromatic carboxylic acids; salicylic, phthalic and thiosalicylic, were found to adsorb to each metal oxide. Salicylic and phthalic acids adsorbed to the metal oxides via bidentate interactions, involving coordination through both carboxylate and substituent groups. Thiosalicylic acid adsorbed to the metal oxides as a bridging bidentate carboxylate with no coordination through the thiol substituent group.
Spectrochim Acta A Mol Biomol Spectrosc 2000 Feb 15
PMID:In situ infrared spectroscopic analysis of the adsorption of aromatic carboxylic acids to TiO2, ZrO2, Al2O3, and Ta2O5 from aqueous solutions. 1079 70

Cytochrome P450 enzyme systems are found throughout nature and are involved in many different, often complex, bioconversions. In the endoplasmic reticulum of the filamentous fungus Aspergillus niger a cytochrome P450 enzyme system is present that is capable of the para-hydroxylation of benzoate. The expression of the two genes encoding the components of this system, the cytochrome P450 gene encoding benzoate para-hydroxylase (bphA) and the gene encoding cytochrome P450 reductase (cprA), is inducible by benzoate. The BPH system was used as a model system to study the mechanisms that result in co-regulation of both components of an eukaryote cytochrome P450 enzyme system. Deletion analysis of the transcription control regions of cprA and bphA resulted in the identification of a region that was involved in benzoate induction of gene expression. The functional competence of the cprA Benzoate Responsive Region thus defined was demonstrated directly by cloning this fragment upstream of a constitutively expressed mini-promoter and analysing expression of the hybrid transcription control region in a lacZ reporter system. Further analysis of cprA gene expression revealed a clear quantitative discrepancy between induction at the protein level (approximately 4-fold) and at the transcription level (> 20-fold). The majority of the transcripts observed after benzoate induction (cprAbeta) were larger then the constitutively expressed cprAalpha transcript. The difference in size between the cprAalpha and cprAbeta transcript is caused by differential promoter use. As the longer cprAbeta transcript carries a small uORF we propose that post-transcriptional regulation of CPR expression underlies the discrepancy in the degree of induction at the protein and transcriptional level. Our results show that regulation of CPR expression is particularly complex, involving regulatory promoter elements, differential promoter use and regulation at the post-transcriptional level.
Mol Gen Genet 2000 May
PMID:Regulation of expression of the Aspergillus niger benzoate para-hydroxylase cytochrome P450 system. 1085 81

The methodology for generating a homology model of the T1 TCR-PbCS-K(d) class I major histocompatibility complex (MHC) class I complex is presented. The resulting model provides a qualitative explanation of the effect of over 50 different mutations in the region of the complementarity determining region (CDR) loops of the T cell receptor (TCR), the peptide and the MHC's alpha(1)/alpha(2) helices. The peptide is modified by an azido benzoic acid photoreactive group, which is part of the epitope recognized by the TCR. The construction of the model makes use of closely related homologs (the A6 TCR-Tax-HLA A2 complex, the 2C TCR, the 14.3.d TCR Vbeta chain, the 1934.4 TCR Valpha chain, and the H-2 K(b)-ovalbumine peptide), ab initio sampling of CDR loops conformations and experimental data to select from the set of possibilities. The model shows a complex arrangement of the CDR3alpha, CDR1beta, CDR2beta and CDR3beta loops that leads to the highly specific recognition of the photoreactive group. The protocol can be applied systematically to a series of related sequences, permitting the analysis at the structural level of the large TCR repertoire specific for a given peptide-MHC complex.
J Mol Biol 2000 Jul 28
PMID:Modeling of the TCR-MHC-peptide complex. 1090 65


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