CYP2E1 active site residues in substrate recognition sequence 5 identified by photoaffinity labeling and homology modeling

Despite its biological importance, our knowledge of active site structure and relevance of critical amino acids in CYP2E1 catalytic processes remain limited. In this study, we identified CYP2E1 active site residues using photoaffinity labeling with 7-azido-4-methylcoumarin (AzMC) coupled with a CYP2E1 homology model. In the absence of light, AzMC was an effective competitor against substrate p-nitrophenol oxidation by CYP2E1. Photoactivation of AzMC led to a concentration-dependent loss in CYP2E1 activity and structural integrity resulting from the modification of both heme and protein. The photo-labeling reaction degraded heme and produced a possible heme adduct. Probe incorporation into the protein occurred at multiple sites within substrate recognition sequence 5 (SRS-5). Based on a CYP2E1 homology model, we hypothesize AzMC labels SRS-5 residues, Leu363, Val364, and Leu368, in the active site. In addition, we propose a series of phenylalanines, especially Phe106, mediate contacts with the coumarin.     

beta-Ketoacyl-[acyl carrier protein] synthase I of Escherichia coli: aspects of the condensation mechanism revealed by analyses of mutations in the active site pocket

beta-Ketoacyl-[acyl carrier protein (ACP)] synthase forms new carbon-carbon bonds in three steps: transfer of an acyl primer from ACP to the enzyme, decarboxylation of the elongating substrate and its condensation with the acyl primer substrate. Six residues of Escherichia coli beta-ketoacyl-ACP synthase I (KAS I) implicated in these reactions were subjected to site-directed mutagenesis. Analyses of the abilities of C163A, C163S, H298A, D306A, E309A, K328A, and H333A to carry out the three reactions lead to the following conclusions. The active site Cys-163 is not required for decarboxylation, whereas His-298 and His-333 are indispensable. Neither of the histidines is essential for increasing the nucleophilicity of Cys-163 to enable transfer of the acyl primer substrate. Maintenance of the structural integrity of the active site by Asp-306 and Glu-309 is required for decarboxylation but not for transfer. One function of Lys-328 occurs very early in catalysis, potentially before transfer. These results in conjunction with structural analyses of substrate complexes have led to a model for KAS I catalysis [Olsen, J. G., Kadziola, A., von Wettstein-Knowles, P., Siggaard-Andersen, M., and Larsen, S. (2001) Structure 9, 233-243]. Another facet of catalysis revealed by the mutant analyses is that the acyl primer transfer activity of beta-ketoacyl-ACP synthase I is inhibited by free ACP at physiological concentrations. Differences in the inhibitory response by individual mutant proteins indicate that interaction of free ACP with Cys-163, Asp-306, Glu-309, Lys-328, and His-333 might form a sensitive regulatory mechanism for the transfer of acyl primers.     

Relationship between ATM and ribosomal protein S6 revealed by the chemical inhibition of Ser/Thr protein phosphatase type 1

The optimal cellular responses to DNA damage are modulated by kinase and phosphatase. The ataxia telangiectasia mutated (ATM) is a Ser/Thr kinase which is the core of the DNA damage signaling apparatus. The Ser/Thr protein phosphatase type 1 (PP1) inhibitor, tautomycetin (TC) and an antibody to the phospho-(S/T)Q sites of the ATM substrate were used to identify the common substrates for PP1 and ATM in regulating the pathway for DNA damage response. Ribosomal protein S6 (RPS6) was first identified as a substrate for PP1 and ATM. The phosphorylation at Ser247 of RPS6 was then significantly decreased by PP1-mediated dephosphorylation immediately after UV irradiation. These results suggest that PP1 specifically dephosphorylated RPS6 at phospho-Ser247 in vivo. In response to DNA damage, ATM activity was finally required for the phosphorylation of RPS6 at Ser247. We propose from these results a novel mechanism for modulating the RPS6 function by PP1 and ATM which regulates cell growth and survival in response to DNA-damage stimuli.     

Altered active site flexibility and a structural metal-binding site in eukaryotic dUTPase: kinetic characterization, folding, and crystallographic studies of the homotrimeric Drosophila enzyme

dUTPase is responsible for preventive DNA repair via exclusion of uracil. Developmental regulation of the Drosophila enzyme is suggested to be involved in thymine-less apoptosis. Here we show that in addition to conserved dUTPase sequence motifs, the gene of Drosophila enzyme codes for a unique Ala-Pro-rich segment. Kinetic and structural analyses of the recombinant protein and a truncation mutant show that the Ala-Pro segment is flexible and has no regulatory role in vitro. The homotrimer enzyme unfolds reversibly as a trimeric entity with a melting temperature of 54 degrees C, 23 degrees C lower than Escherichia coli dUTPase. In contrast to the bacterial enzyme, Mg(2+) binding modulates conformation of fly dUTPase, as identified by spectroscopy and by increment in melting temperature. A single well folded, but inactive, homotrimeric core domain is generated through three distinct steps of limited trypsinolysis. In fly, but not in bacterial dUTPase, binding of the product dUMP induces protection against proteolysis at the tryptic site reflecting formation of the catalytically competent closed conformer. Crystallographic analysis argues for the presence of a stable monomer of Drosophila dUTPase in crystal phase. The significant differences between prototypes of eukaryotic and prokaryotic dUTPases with respect to conformational flexibility of the active site, substrate specificity, metal ion binding, and oligomerization in the crystal phase are consistent with alteration of the catalytic mechanism and hydropathy of subunit interfaces.     

Structural studies,homology modeling and molecular docking of novel non-competitive antagonists of GluK1/GluK2 receptors

Non-competitive ligands of kainate receptors have focused significant attention as medicinal compounds because they seem to be better tolerated than competitive antagonists and uncompetitive blocker of these receptors. Here we present structural studies (X-ray structure determination, NMR and MS spectra) of novel indole-derived non-competitive antagonists of GluK1/GluK2 receptors, homology models of GluK1 and GluK2 receptors based on novel AMPA receptor template as well as molecular docking of ligands to their molecular targets. We find that the allosteric site is in the receptor transduction domain, in one receptor subunit, not between the two subunits as it was indicated by our earlier studies.     

Comparative kinetic studies on the L-type pyruvate kinase from rat liver and the enzyme phosphorylated by cyclic 3', 5'-AMP-stimulated protein kinase.

The kinetics of rat liver L-type pyruvate kinase (EC 2.7.1.40), phosphorylated with cyclic AMP-stimulated protein kinase from the same source, and the unphosphorylated enzyme have been compared. The effects of pH and various concentrations of substrates, Mg2+, K+ and modifiers were studied. In the absence of fructose 1, 6-diphosphate at pH 7.3, the phosphorylated pyruvate kinase appeared to have a lower affinity for phosphoenolpyruvate (K0.5=0.8 mM) than the unphosphorylated enzyme (K0.5=0.3 mM). The enzyme activity vs. phosphoenolpyruvate concentration curve was more sigmoidal for the phosphorylated enzyme with a Hill coefficient of 2.6 compared to 1.6 for the unphosphorylated enzyme. Fructose 1, 6-diphosphate increased the apparent affinity of both enzyme forms for phosphoenolpyruvate. At saturating concentrations of this activator, the kinetics of both enzyme forms were transformed to approximately the same hyperbolic curve, with a Hill coefficient of 1.0 and K0.5 of about 0.04 mM for phosphoenolpyruvate. The apparent affinity of the enzyme for fructose 1, 6-diphosphate was high at 0.2 mM phosphoenolpyruvate with a K0.5=0.06 muM for the unphosphorylated pyruvate kinase and 0.13 muM for the phosphorylated enzyme. However, in the presence of 0.5 mM alanine plus 1.5 mM ATP, a higher fructose 1, 6-diphosphate concentration was needed for activation, with K0.5 of 0.4 muM for the unphosphorylated enzyme and of 1.4 muM for the phosphorylated enzyme. The results obtained strongly indicate that phosphorylation of pyruvate kinase may also inhibit the enzyme in vivo. Such an inhibition should be important during gluconeogenesis.     

Potent and selective inhibition of the tumor marker AKR1B10 by bisdemethoxycurcumin: Probing the active site of the enzyme with molecular modeling and site-directed mutagenesis

A human member of the aldo-keto reductase (AKR) superfamily, AKR1B10, shares high sequence identity with aldose reductase (AR), and was recently identified as a therapeutic target in the treatment of several types of cancer. We have compared the inhibitory effects of plant components on recombinant AKR1B10 and AR. AKR1B10 was inhibited by curcuminoids, magnolol, honokiol and resveratrol, with IC₅₀ values of 0.06-5 μM, which were lower than their values for AR. Among them, bisdemethoxycurcumin was the most potent competitive inhibitor (K_i = 22 nM) with the highest selectivity (85-fold versus AR), and acted as an effective inhibitor in cellular level. In contrast, demethoxycurcumin and curcumin showed >3-fold less potency and selectivity. Molecular docking studies of the curcuminoids in the AKR1B10-NADP⁺ complex and site-directed mutagenesis of the putative binding residues suggest that Gln114, Val301 and Gln303 are important for determining the inhibitory potency and selectivity of the curcuminoids.     

Cyclopropane fatty acid synthase of Escherichia coli: deduced amino acid sequence, purification, and studies of the enzyme active site.

Cyclopropane fatty acid (CFA) synthase of Escherichia coli catalyzes a modification of the acyl chains of phospholipid bilayers. We report (i) identification of the CFA synthase protein, (ii) overproduction (> 600-fold) and purification to essential homogeneity of the enzyme, and (iii) the amino acid sequence of CFA synthase as deduced from the nucleotide sequence of the cfa gene. CFA synthase was overproduced by use of the T7 promoter/RNA polymerase system under closely defined conditions. The enzyme was readily purified by a two-step procedure requiring only ammonium sulfate fractionation and binding to phospholipid vesicles followed by flotation in sucrose density gradients. The deduced amino acid sequence predicts a protein of 43,913 Da (382 residues) that lacks long hydrophobic segments. The CFA synthase sequence has no significant similarity to known proteins except for sequences found in other enzymes that utilize S-adenosyl-L-methionine. We also report inhibitor studies of the enzyme active site.     

Characterization of a sperm-specific nuclear autoantigenic protein. I. Complete sequence and homology with the Xenopus protein, N1/N2

In our studies on specific sperm proteins that function in fertilization, an autoantigenic, postacrosomal sperm protein has been found to originate in the testis as a nuclear-associated protein. This nuclear autoantigenic sperm protein (NASP) contains a C-terminal nuclear translocation signal and has structural similarities to the lamins and other nuclear proteins; and its 2.5 kb mRNA is apparently tissue-, but not species-, specific. DNA clones from a rabbit testis cDNA library and a rabbit genomic library were sequenced in order to characterize NASP. The polyadenylated mRNA has 39 bases of 5' untranslated sequence, an open reading frame of 2043 bases encoding 680 amino acids, and a 104 base 3' untranslated region (2,186). The encoded polypeptide has a calculated molecular weight of 73,533 and a pI = 4.06, containing 25% acidic residues. One clone (R1.2) expressing the C-terminal 446 amino acids was used to express a fusion protein. The expressed R1.2/beta-galactosidase fusion protein was found to be autoantigenic. Secondary structure predictions for NASP showed that 69% of the molecule had a high probability of forming alpha-helices and that several alpha-helical regions had a characteristic repeating heptad pattern that in the intermediate filaments and nuclear lamins is involved in coiled-coil interactions with other molecules. In addition to the nuclear translocation signal common to many nuclear proteins, NASP also showed homology with the Xenopus histone-binding protein, N1/N2.     

Probing the mechanism of hamster arylamine N-acetyltransferase 2 acetylation by active site modification, site-directed mutagenesis, and pre-steady state and steady state kinetic studies

Arylamine N-acetyltransferases (NATs) catalyze an acetyl group transfer from acetyl coenzyme A (AcCoA) to arylamines, hydrazines, and their N-hydroxylated arylamine metabolites. The recently determined three-dimensional structures of prokaryotic NATs have revealed a cysteine protease-like Cys-His-Asp catalytic triad, which resides in a deep and hydrophobic pocket. This catalytic triad is strictly conserved across all known NATs, including hamster NAT2 (Cys-68, His-107, and Asp-122). Treatment of NAT2 with either iodoacetamide (IAM) or bromoacetamide (BAM) at neutral pH rapidly inactivated the enzyme with second-order rate constants of 802.7 +/- 4.0 and 426.9 +/- 21.0 M(-1) s(-1), respectively. MALDI-TOF and ESI mass spectral analysis established that Cys-68 is the only site of alkylation by IAM. Unlike the case for cysteine proteases, no significant inactivation was observed with either iodoacetic acid (IAA) or bromoacetic acid (BAA). Pre-steady state and steady state kinetic analysis with p-nitrophenyl acetate (PNPA) and NAT2 revealed a single-exponential curve for the acetylation step with a second-order rate constant of (1.4 +/- 0.05) x 10(5) M(-1) s(-1), followed by a slow linear rate of (7.85 +/- 0.65) x 10(-3) s(-1) for the deacetylation step. Studies of the pH dependence of the rate of inactivation with IAM and the rate of acetylation with PNPA revealed similar pK(a)(1) values of 5.23 +/- 0.09 and 5.16 +/- 0.04, respectively, and pK(a)(2) values of 6.95 +/- 0.27 and 6.79 +/- 0.25, respectively. Both rates reached their maximum values at pH 6.4 and decreased by only 30% at pH 9.0. Kinetic studies in the presence of D(2)O revealed a large inverse solvent isotope effect on both inactivation and acetylation of NAT2 [k(H)(inact)/k(D)(inact) = 0.65 +/- 0.02 and (k(2)/K(m)(acetyl))(H)/(k(2)/K(m)(acetyl))(D) = 0.60 +/- 0.03], which were found to be identical to the fractionation factors (Phi) derived from proton inventory studies of the rate of acetylation at pL 6.4 and 8.0. Substitution of the catalytic triad Asp-122 with either alanine or asparagine resulted in the complete loss of protein structural integrity and catalytic activity. From these results, it can be concluded that the catalytic mechanism of NAT2 depends on the formation of a thiolate-imidazolium ion pair (Cys-S(-)-His-ImH(+)). However, in contrast to the case with cysteine proteases, a pH-dependent protein conformational change is likely responsible for the second pK(a), and not deprotonation of the thiolate-imidazolium ion. In addition, substitutions of the triad aspartate are not tolerated. The enzyme appears, therefore, to be engineered to rapidly form a stable acetylated species poised to react with an arylamine substrate.     

Structure of the human glycogen-associated protein phosphatase 1 regulatory subunit hGM: homology modeling revealed an (alpha/beta)8-barrel-like fold in the multidomain protein

Protein phosphatase 1 (PP1) is widely distributed among tissues and species and acts as a regulator of many important cellular processes. By targeting the catalytic part of PP1 (PP1C) toward particular loci and substrates, regulatory subunits constitute key elements conferring specificity to the holoenzyme. Here, we report the identification of an (alpha/beta)8-barrel-like structure within the N-ter stretch of the human PP1 regulatory subunit hGM, which is part of the family of diverse proteins associated with glycogen metabolism. Protein homology modeling gave rise to a three-dimensional (3D) model for the 381 N-ter residue stretch of hGM, based on sequence similarity with Streptomyces olivochromogenes xylose isomerase, identified by using FASTA. The alignment was subsequently extended by using hydrophobic cluster analysis. The homology-derived model includes the putative glycogen binding area located within the 142-230 domain of hGM as well as a structural characterization of the PP1C interacting domain (segment 51-67). Refinement of the latter by molecular dynamics afforded a topology that is in agreement with previous X-ray studies (Egloff et al., 1997). Finite difference Poisson-Boltzmann calculations performed on the interacting domains of PP1C and hGM confirm the complementarity of the local electrostatic potentials of the two partners. This work highlights the presence of a conserved fold among distant species (mammalian, Caenorhabditis elegans, yeast) and, thus, emphasizes the involvement of PP1 in crucial basic cellular functions.     

The 3D structure of the defense-related rice protein Pir7b predicted by homology modeling and ligand binding studies

To better understand the ligand-binding mechanism of protein Pir7b, important part in detoxification of a pathogen-derived compound against Pyricularia oryzae, a 3D structure model of protein Pir7b was constructed based on the structure of the template SABP2. Three substrates were docking to this protein, two of them were proved to be active, and some critical residues are identified, which had not been confirmed by the experiments. His87 and Leu17 considered as ‘oxyanion hole’ contribute to initiating the Ser86 nucleophilic attack. Gln187 and Asp139 can form hydrogen bonds with the anilid group to maintain the active binding orientation with the substrates. The docking model can well interpret the specificity of protein Pir7b towards the anilid moiety of the substrates and provide valuable structure information about the ligand binding to protein Pir7b. Figure Ligand binding analysis based on the refined Pir7b model. Magenta dash line, hydrogen bond; Red dash line, distance label. (a) Docking of 2-naphthol AS-acetate to Pir7b model. A 3D figure of 2-naphthol AS-acetate-Pir7b complex is also attached (b) Docking of 2-naphthol AS-2-chlor-propionate to Pir7b model. (c) Docking of 2-naphthol-acetate to Pir7b model.     

Acetyl-CoA-carboxylase: modification of ATP-binding site of the active center by 2',3'-dialdehyde derivative of ATP

The interaction of rat liver acetyl-CoA carboxylase with a 2',3'-dialdehyde derivative of ATP (oATP) has been studied. The degree of the enzyme inactivation has been found to depend on the oATP concentration and the incubation time. ATP was the only reaction substrate which provided protection from inactivation. Acetyl-CoA did not affect inactivation, while HCO3- accelerated the process. Ki values for oATP in the absence and the presence of HCO3- were 0.35 +/- 0.04 and 0.5 +/- 0.06 mM, and those of the modification constant (k) were 0.11 and 0.26 min-1, respectively. oATP completely inhibited the reaction of [14C]ADP in equilibrium ATP exchange, whereas produced actually no effect on [14C]acetyl-CoA equilibrium with malonyl-CoA exchange. Incorporation of about one equivalent of [3H]oATP per acetyl-CoA carboxylase subunit has been shown. No restoration of the modified enzyme activity has been observed in Tris or beta-mercaptoethanol containing buffers, and treatment with NaB[3H]4 has not led to 3H incorporation. The modification process involves elimination of the triphosphate chain of oATP. The results obtained indicate the affinity character of oATP-mediated modification of acetyl-CoA carboxylase. The reagent apparently interacts selectively with the epsilon-amino group of lysine in the ATP-binding site to form a morpholine-like structure.     

The phosphoenolpyruvate: methyl-α-D-glucoside phosphotransferase system in Bacillus subtilis Marburg: kinetic studies of enzyme II and evidence for a phosphoryl enzyme II intermediate

The Enzyme II complex catalyzing the phosphoryl transfer from P-HPr to sugar in the inducible methyl-α-D-glucoside: phosphotransferase system in Bacillus subtilis acts according to a ping-pong mechanism, implying a phosphorylated Enzyme II intermediate. This result is supported by the demonstration of a specific transphosphorylation between [¹⁴C] αMG and glucose-6-phosphate in the presence of an induced Enzyme II preparation.     

The targets of 2,2',5,5'-tetrachlorobiphenyl in the respiratory chain of rat liver mitochondria revealed by modular kinetic analysis

The response of the respiratory subsystem of oxidative phosphorylation to the environmental pollutant, 2,2',5,5'-tetrachlorobiphenyl (2,2',5,5'-TCB) was investigated by modular kinetic approach. The effects of 20 M 2,2',5,5'-TCB on the activity of the respiratory chain modules in rat liver mitochondria oxidizing succinate (+ rotenone) in state 3 were assessed. The toxin inhibited the rate of respiration by 23%. Analysis around cytochrome c revealed that 2,2',5,5'-TCB inhibited both cytochrome c-oxidizing and - reducing modules. The toxin inhibited also CoQ-oxidizing module, however it did not affect the kinetics of CoQ-reducing module. Taken together, these data indicated that 2,2',5,5'-TCB inhibited cytochrome bc₁ but had no effect on succinate dehydrogenase.     

Probing conformations of the glycerol backbones of triacyglycerols in the active site of lipase by 1,2-cyclopentane-carbamates: The meso effect for the enzyme inhibition

The aim of this study is to probe the glycerol backbone conformation of the substrate (or inhibitor) in the active site of Pseudomonas species lipase by the 1,2-cyclopentandiol analogues of the ethylene glycerol carbamate inhibitors. Cyclopentane-carbamates, cis-1,2-di-N-n- butylcarbamyl-cyclopentane (1) and trans-1,2-di-N-n-butylcarbamyl-cyclopentane (2), are the conformationally constrained analogues of 1,2-di-N-n-butylcarbamyl ethane (3). All carbamates are synthesized and characterized as the pseudo-substrate inhibitors of the enzyme. Cis-cyclopentane-di-carbamate (1) is a more potent inhibitor than both ethane-di-carbamate (3) and trans-cyclopentane-di-carbamate (2) probably because the glycerol backbone conformations of cis-cyclopentane-di-carbamate (1) are constrained by the cyclopentane ring and cis-cyclopentane-di-cabamate (1) is a meso compound but trans-cyclopentane-di-carbamate (2) is a racemate.     

Variation in antiviral 2',5'-oligoadenylate synthetase (2'5'AS) enzyme activity is controlled by a single-nucleotide polymorphism at a splice-acceptor site in the OAS1 gene

It is likely that human genetic differences mediate susceptibility to viral infection and virus-triggered disorders. OAS genes encoding the antiviral enzyme 2',5'-oligoadenylate synthetase (2'5'AS) are critical components of the innate immune response to viruses. This enzyme uses adenosine triphosphate in 2'-specific nucleotidyl transfer reactions to synthesize 2',5'-oligoadenylates, which activate latent ribonuclease, resulting in degradation of viral RNA and inhibition of virus replication. We showed elsewhere that constitutive (basal) activity of 2'5'AS is correlated with virus-stimulated activity. In the present study, we asked whether constitutive activity is genetically determined and, if so, by which variants. Analysis of 83 families containing two parents and two children demonstrated significant correlations between basal activity in parent-child pairs (P<.0001) and sibling pairs (P=.0044), but not spousal pairs, suggesting strong genetic control of basal activity. We next analyzed association between basal activity and 15 markers across the OAS gene cluster. Significant association was detected at multiple markers, the strongest being at an A/G single-nucleotide polymorphism at the exon 7 splice-acceptor site (AG or AA) of the OAS1 gene. At this unusual polymorphism, allele G had a higher gene frequency in persons with high enzyme activity than in those with low enzyme activity (0.44 vs. 0.20; P=3 x 10(-11)). Enzyme activity varied in a dose-dependent manner across the GG, GA, and AA genotypes (tested by analysis of variance; P=1 x 10(-14)). Allele G generates the previously described p46 enzyme isoform, whereas allele A ablates the splice site and generates a dual-function antiviral/proapoptotic p48 isoform and a novel p52 isoform. This genetic polymorphism makes OAS1 an excellent candidate for a human gene that influences host susceptibility to viral infection.