Identification of active site residues in the "GyrA" half of yeast DNA topoisomerase II

Site-directed mutagenesis was carried out at 10 highly conserved polar residues within the C-terminal half of yeast DNA topoisomerase II, which corresponds to the A subunit of bacterial DNA gyrase, to identify amino acid side chains that augment the active site tyrosine Tyr-782 in the breakage and rejoining of DNA strands. Complementation tests show that alanine substitution at Arg-690, Asp-697, Lys-700, Arg-704, or Arg-781, but not at His-735, His-736, Glu-738, Gln-750, or Asn-828, inactivates the enzyme in vivo. Measurements of DNA relaxation and cleavage by purified mutant enzymes show that these activities are abolished in the R690A mutant and are much reduced in the mutants D697A, K700A, R704A, and R781A. When a Y782F polypeptide with a phenylalanine substituting for the active site tyrosine was expressed in cells that also express the R690A polypeptide, the resulting heterodimeric yeast DNA topoisomerase II was found to nick plasmid DNA. Thus in a dimeric wild-type enzyme, Tyr-782 in one protomer and Arg-690 in the other cooperate in trans in the catalysis of DNA cleavage. For the residues D697A, K700A, R704A, and R781A, their locations in the crystal structures of type II DNA topoisomerase fragments suggest that Arg-781 and Lys-700 might be involved in anchoring the 5' and 3' sides of the broken DNA, respectively, and the roles of Asp-697 and Arg-704 are probably less direct.     

Identification of a firefly luciferase active site peptide using a benzophenone-based photooxidation reagent

Firefly luciferase catalyzes the highly efficient emission of yellow-green light from substrate luciferin by a series of reactions that require MgATP and molecular oxygen. We prepared 2-(4-benzoylphenyl)thiazole-4-carboxylic acid (BPTC), a novel benzophenone-based substrate analog, intending to use it in photoaffinity labeling studies to probe the luciferase active site. Instead, we found that while BPTC was a potent photoinactivating reagent for firefly luciferase, it was not a photoaffinity labeling agent. Using proteolysis, reverse phase high-performance liquid chromatography, tandem high performance liquid chromatography-electrospray ionization mass spectrometry, and Edman sequencing, we identified a single luciferase peptide, 244HHGF247, the degradation of which was directly correlated to luciferase photoinactivation. Results of enzyme kinetics and related studies were consistent with this peptide being at or near the luciferin binding site. Further, peptide model studies and additional investigations on the nature of the photoinactivation process strongly suggested that BPTC catalyzed the formation of singlet oxygen at the active site of the enzyme. We describe here an uncommon example of active site-directed photooxidation of an enzyme by singlet oxygen.     

Localization and in vitro mutagenesis of the active site in the Saccharomyces cerevisiae mRNA capping enzyme

The yeast mRNA capping enzyme is composed of 52 (alpha) and 80 kDa (beta) polypeptides, which are responsible for its mRNA guanylyltransferase and RNA 5'-triphosphatase activities, respectively. We isolated the gene encoding the alpha subunit (CEG1) and showed that CEG1 is essential for yeast cell growth [Shibagaki et al., (1992) J. Biol. Chem. 267, 9521-9528]. In this study, CEG1 was expressed in Escherichia coli and the alpha subunit protein was purified to near homogeneity. A [32P]GMP-bound tryptic peptide derived from the recombinant enzyme-[32P]GMP covalent reaction intermediate was converted to a [32P]phosphoryl-peptide through periodate oxidation followed by beta-elimination. Hydrolysis of the [32P]phosphoryl-peptide with alkali resulted in [32P]N epsilon-phospholysine as the only phosphoamino acid, indicating that GMP in the enzyme-GMP complex is bound to a lysine residue via a phosphoamide linkage. Microsequencing of the [32P]GMP-peptide showed that the GMP binding site was located in the region between amino acids 60 and 75, which contained an internal trypsin-resistant lysine at position 70. CEG1 was subjected to site-directed mutagenesis and the mutant proteins were expressed in E. coli. Substitution of His or Ile for Lys70 entirely abolished the enzyme-GMP formation activity, and this mutation was lethal to yeast in vivo, supporting the notion that the active site in the alpha subunit is located at Lys70. Replacement of Lys70 with Arg reduced the ability to form the enzyme-GMP complex; however, yeast cells bearing this allele were not viable. A series of mutations, including 8 amino acid replacements and 3 insertions, near the active site (Lys70-Thr-Asp-Gly motif) were also introduced and the mutant polypeptides were examined for catalytic activity in vitro as well as yeast cell viability in vivo. There was a good correlation between the in vitro and in vivo functions of the mutant proteins, except when Asp72 was replaced with Glu, which allowed formation of the enzyme-GMP complex but failed to support cell growth. The results with Lys70 to Arg and Asp72 to Glu substitutions indicated that guanylyltransfer to RNA and/or additional roles besides cap formation per se are impaired in these mutant proteins.     

Identification of a Gialpha binding site on type V adenylyl cyclase

The stimulatory G protein alpha subunit Gsalpha binds within a cleft in adenylyl cyclase formed by the alpha1-alpha2 and alpha3-beta4 loops of the C2 domain. The pseudosymmetry of the C1 and C2 domains of adenylyl cyclase suggests that the homologous inhibitory alpha subunit Gialpha could bind to the analogous cleft within C1. We demonstrate that myristoylated guanosine 5'-3-O-(thio)triphosphate-Gialpha1 forms a stable complex with the C1 (but not the C2) domain of type V adenylyl cyclase. Mutagenesis of the membrane-bound enzyme identified residues whose alteration either increased or substantially decreased the IC50 for inhibition by Gialpha1. These mutations suggest binding of Gialpha within the cleft formed by the alpha2 and alpha3 helices of C1, analogous to the Gsalpha binding site in C2. Adenylyl cyclase activity reconstituted by mixture of the C1 and C2 domains of type V adenylyl cyclase was also inhibited by Gialpha. The C1b domain of the type V enzyme contributed to affinity for Gialpha, but the source of C2 had little effect. Mutations in this soluble system faithfully reflected the phenotypes observed with the membrane-bound enzyme. The pseudosymmetrical structure of adenylyl cyclase permits bidirectional regulation of activity by homologous G protein alpha subunits.     

Rapid binding of a cationic active site inhibitor to wild type and mutant mouse acetylcholinesterase: Brownian dynamics simulation including diffusion in the active site gorge

It is known that anionic surface residues play a role in the long-range electrostatic attraction between acetylcholinesterase and cationic ligands. In our current investigation, we show that anionic residues also play an important role in the behavior of the ligand within the active site gorge of acetylcholinesterase. Negatively charged residues near the gorge opening not only attract positively charged ligands from solution to the enzyme, but can also restrict the motion of the ligand once it is inside of the gorge. We use Brownian dynamics techniques to calculate the rate constant kon, for wild type and mutant acetylcholinesterase with a positively charged ligand. These calculations are performed by allowing the ligand to diffuse within the active site gorge. This is an extension of previously reported work in which a ligand was allowed to diffuse only to the enzyme surface. By setting the reaction criteria for the ligand closer to the active site, better agreement with experimental data is obtained. Although a number of residues influence the movement of the ligand within the gorge, Asp74 is shown to play a particularly important role in this function. Asp74 traps the ligand within the gorge, and in this way helps to ensure a reaction.     

Identification and precursor frequency analysis of a common T cell epitope motif in mitochondrial autoantigens in primary biliary cirrhosis

The immunodominant antimitochondrial antibody response in patients with primary biliary cirrhosis (PBC) is directed against the E2 component of the pyruvate dehydrogenase complex (PDC-E2). Based on our earlier observations regarding peripheral blood mononuclear cell (PBMC) T cell epitopes, we reasoned that a comparative analysis of the precursor frequencies of PDC-E2 163-176-specific T cells isolated from PBMC, regional hepatic lymph nodes, and from the liver of PBC patients would provide insight regarding the role of T cells in PBC. Results showed a disease-specific 100-150-fold increase in the precursor frequency of PDC-E2 163-176-specific T cells in the hilar lymph nodes and liver when compared with PBMC from PBC patients. Interestingly, autoreactive T cells and autoantibodies from PBC patients both recognize the same dominant epitope. In addition, we demonstrated cross-reactivity of PDC-E2 peptide 163-176-specific T cell clones with PDC-E2 peptide 36-49 and OGDC-E2 peptide 100-113 thereby identifying a common T cell epitope "motif" ExETDK. The peptide 163-176-specific T cell clones also reacted with purified native PDC-E2, suggesting that this epitope is not a cryptic determinant. These data provide evidence for a major role for PDC-E2 peptide 163-176 and/or peptides bearing a similar motif in the pathogenesis of PBC.     

Identification of the essential cysteinyl residue located in the active site of human phenylethanolamine N-methyltransferase

Rabbit (Oryctolagus cuniculus) red cell and tissue acid phosphatases were studied by means of horizontal starch gel electrophoresis and isoelectric focusing followed by enzyme blotting. Red cell acid phosphatase 1 (ACP1) is monomorphic while tissue acid phosphatase 2 (ACP2) is polymorphic in a wild rabbit population, with two alleles: ACP2*1 (0.96) and ACP2*2 (0.04). A third locus homologous of human acid phosphatase 3 (ACP3) is characterized by the presence of three alleles (ACP3*1, ACP3*2 and ACP3*3). ACP3*1 is the most common allele and was detected in all populations, ACP3*2 was found in domestic breeds and in a wild population from Southern France, whereas ACP3*3 is typical of Portuguese wild rabbits. The geographical distribution of ACP3*2 and ACP3*3 is in agreement with the subspecific level of differentiation of the rabbit species in O. cuniculus cuniculus and O. c. algirus. The comparative study of the acid phosphatase activity in red cells of several mammalian species, including humans, suggests that ACP3 activity in erythrocytes exists only in rabbit.     

The role of the active site cysteine in catalysis by type 1 iodothyronine deiodinase

Type 1 iodothyronine deiodinase (deiodinase 1) is a selenoenzyme that converts the prohormone T4 to the active thyroid hormone T3 by outer ring deiodination or to the inactive metabolite rT3 by inner ring deiodination. Although selenocysteine has been demonstrated to be essential for the biochemical profile of deiodinase 1, the role of a highly conserved, active site cysteine (C124 in rat deiodinase 1) has not been defined. The present studies examined the effects of a Cys124Ala mutation on rat deiodinase 1 enzymatic function and substrate affinity. At a constant 10-mM concentration of dithiothreitol (DTT), the C124A mutant demonstrated a 2-fold lower apparent maximal velocity (Vmax) and Km for rT3 (KmrT3) than the wild type for outer ring deiodination, whereas the Vmax/Km ratio was unchanged. Similarly, the apparent Vmax and KmT3 sulfate for inner ring deiodination were 2-fold lower in the C124A mutant relative to those in the wild type, with no change in the Vmax/Km ratio. The C124A mutant exhibited ping-pong kinetics in the presence of DTT, and substitution of the active site cysteine increased the KmDTT by 14-fold relative to that of the wild-type enzyme, with no significant effects on KmrT3 or Vmax. The C124A mutant was inhibited by propylthiouracil in an uncompetitive fashion and exhibited a 2-fold increase in K(i)propylthiouracil compared with that of the wild type. KmrT3 was also reduced for the C124A mutant when 5 mM reduced glutathione, a potential physiological monothiol cosubstrate, was used in outer ring deiodination assays. These results demonstrate that thiol cosubstrate interactions with C124 in type 1 deiodinase play an important role in enhancing catalytic efficiency for both outer and inner ring deiodination.     

Ontogenetic dissociation of two classes of habituation.

Determined the rates of decrement of 2 classes of response (an elicited startle reflex and emitted exploratory behavior) in rats of 2 different ages (15 and 36 days). Ss were 31 Sprague-Dawley albino rats. The rate of decrement in the startle reflex was not clearly differentiated as a function of age. In contrast, there was no evidence of habituation of exploration in the younger Ss, whereas older Ss uniformly showed profound response decrements. This ontogenetic dissociation of the 2 instances of response decrement indicates that accounts of both instances in terms of a common process called habituation may be unwarranted. In addition, these data, in conjunction with earlier findings, indirectly support the possibility that reflex decrements may be relatively more dependent on brain serotonin, whereas decrements in exploration may be more dependent on normal cholinergic activity in brain. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Cholinergic sites in skeletal muscle. II. Interaction of an agonist and two antagonists with the acetylcholine site

The equilibrium interactions of alpha-bungarotoxin, d-tubocurarine, and carbamylcholine with junctional and extrajunctional skeletal muscle acetylcholine receptors were examined. d-Tubocurarine is a competitive inhibitor of the bindings of alpha-bungarotoxin to the acetylcholine receptor. No substantive difference was observed in the association of d-tubocurarine with the junctional and extrajunctional receptors. In contrast, the carbamylcholine inhibition of toxin binding is not competitive. The data indicate that either the single set of alpha-bungarotoxin and d-tubocurarine bindings sites contains two subsets of carbamylcholine sites or that the carbamylcholine binds in a cooperative manner to a single set of sites. In addition, the affinity of carbamylcholine for extrajunctional receptors may be higher than the affinity for junctional receptors.     

Localization of the major NF-kappaB-activating site and the sole TRAF3 binding site of LMP-1 defines two distinct signaling motifs

The TRAF3 molecule interacts with the cytoplasmic carboxyl terminus (COOH terminus) of the Epstein-Barr virus-encoded oncogene LMP-1. NF-kappaB activation is a downstream signaling event of tumor necrosis factor receptor-associated factor (TRAF) molecules in other signaling systems (CD40 for example) and is an event caused by LMP-1 expression. One region capable of TRAF3 interaction in LMP-1 is the membrane-proximal 45 amino acids (188-242) of the COOH terminus. We show that this region contains the only site for binding of TRAF3 in the 200-amino acid COOH terminus of LMP-1. The site also binds TRAF2 and TRAF5, but not TRAF6. TRAF3 binds to critical residues localized between amino acids 196 and 212 (HHDDSLPHPQQATDDSG), including the PXQX(T/S) motif, that share limited identity to the CD40 receptor TRAF binding site (TAAPVQETL). Mutation of critical residues in the TRAF3 binding site of LMP-1 that prevents binding of TRAF2, TRAF3, and TRAF5 does not affect NF-kappaB-activating potential. Deletion mapping localized the major NF-kappaB activating region of LMP-1 to critical residues in the distal 4 amino acids of the COOH terminus (383-386). Therefore, TRAF3 binding and NF-kappaB activation occur through two separate motifs at opposite ends of the LMP-1 COOH-terminal sequence.     

Identification of two classes of lipid molecule binding sites on the microsomal triglyceride transfer protein

The gene for the microsomal triglyceride transfer protein (MTP) is defective in subjects with the genetic disease abetalipoproteinemia, indicating that MTP is essential for the assembly of apolipoprotein B containing lipoproteins. In vitro, MTP is a lipid molecule binding protein that catalyzes lipid transport between membranes by a shuttle mechanism. In this study, the lipid binding properties of MTP were examined. MTP was incubated with donor phosphatidylcholine vesicles of varying neutral lipid composition. MTP was subsequently reisolated by ultracentrifugation, and MTP-bound lipid was quantitated. When the triolein content of the vesicles was increased up to 4 mol %, neutral lipid binding to MTP increased proportionately, while phosphatidylcholine binding appeared to remain constant around two molecules per MTP. Using phosphatidylcholine emulsions containing 60 mol % triolein as the donor particles resulted in only a slight increase in triolein binding to MTP. The highest triolein:MTP ratio observed was (0.20-0.25):1. Differences in the neutral and phospholipid binding properties of MTP were observed by measuring the transport of lipid from MTP to acceptor vesicles. Transport of triolein was rapid and complete, while phosphatidylcholine transport was biphasic, containing rapid and slow phases. These results indicated that MTP contains more than one class of lipid molecule binding site. Measurements of fluorescent lipid transport from donor vesicles to MTP supported this hypothesis. The transport of pyrene-labeled triglyceride from donor particles to MTP was rapid, while phosphatidylcholine transfer had fast and slow phases. From these data, we propose that MTP contains at least two distinct classes of lipid molecule binding sites that differ in function. The fast site or sites are responsible for lipid transport.     

Localization of carboxy-terminal type II procollagen peptide (pCOL-II-C) and type II collagen in the repair tissue of full-thickness articular cartilage defect

Apoptosis and aging share common mechanisms in oxidative stress and mitochondrial involvement. Treatment of cultured neuroblastoma cells with a radical initiator induced apoptosis; raise in hydrogen peroxide and release of cytochrome c from mitochondria preceded collapse of mitochondrial potential and cell death. In rat hepatocytes treated with adriamycin incubation with exogenous Coenzyme Q10 counteracted the drug-induced increase of hydrogen peroxide and the fall of the mitochondrial potential, thus demonstrating the quinone antioxidant effect. Complex I activity and its rotenone sensitivity decreased in brain cortex non-synaptic mitochondria from old rats; a 5 kb mitochondrial DNA deletion was found only in the old rats. A similar behavior was found in human platelets from old individuals. The postulated energy decline was confirmed by the inhibitor sensitivities of platelet aggregation and lactate production. The lack of the 5 kb deletion in platelets throws doubts on mitochondrial DNA lesions as the only causes of mitochondrial dysfunction in aging.     

Allele-specific late replication and fragility of the most active common fragile site, FRA3B

FRA3B at 3p14.2 is the most active of the common fragile sites in the human genome and is expressed when cells are exposed to the DNA replication inhibitor, aphidicolin. Several lines of evidence suggest that fragile sites are regions of late replication. To elucidate the relationship between the timing of replication across the FRA3B region and its corresponding fragility, we labeled cells with 5-bromo-2'-deoxyuridine (BrdU) and adopted an immunofluorescent procedure to visualize late replicating DNA (BrdU-substituted DNA) in metaphase chromosomes. We also chose 21 markers along the FRA3B region and analyzed the timing of replication using BrdU-labeled DNA from different stages of the cell cycle sorted by flow cytometry. Our results show that there are two distinct alleles that replicate at different stages in the cell cycle and that breaks/gaps preferentially occurred on the chromosome 3 with the late replication allele. These results provide direct evidence that allele-specific late replication is involved in the fragility of the most active common fragile site, FRA3B.     

Identification of a beta-lactoglobulin lactosylation site

In various animal models of cerebral hypoxia-ischemia, it is not clear whether neuronal apoptosis results from hypoxia alone or whether other factors mediate this process. We hypothesized that (1) hypoxia alone can induce neuronal apoptosis, (2) hypoxic severity alters the time course of neuronal apoptosis, (3) hypoxia increases neuronal p53, and this increase in p53 is critical for neuronal apoptosis. Embryonic neocortical neurons cultured for 7-10 days were placed in an incubator with levels set at 0.1%, 1%, and 3% O2 and were removed at 24-h intervals for study. Under all hypoxic conditions, observed changes in cellular morphology and DNA fragmentation, detected by the TUNEL method and gel electrophoresis, were consistent with apoptosis. These alterations were seen after a shorter period with increasing hypoxic severity. Immunoblot analysis revealed an increase in p53 protein in hypoxia-exposed neurons. Analysis of immunofluorescence-stained neurons revealed increases in p53 with increased duration and severity of hypoxia. Antisense oligonucleotides for p53 significantly increased the number of surviving neurons during hypoxic exposure. We conclude that hypoxia-induced neuronal apoptosis is, in part, a p53-dependent process whose time course is influenced by hypoxic severity and duration.     

Identification of the active site serine of penicillin-binding protein 2a from methicillin-resistant Staphylococcus aureus by electrospray mass spectrometry

Penicillin-binding protein 2a (PBP2a), a high molecular mass PBP, is the primary enzyme responsible for the beta-lactam resistance in methicillin-resistant Staphylococcus aureus (MRSA). Inhibition of a PBP such as PBP2a by beta-lactams is due to covalent modification of an active site serine residue. Based on the sequence alignment with well studied beta-lactamases, DD-carboxypeptidases and other high molecular mass PBPs, the serine of a tetrad S403XXK in PBP2a was tentatively identified as the penicillin-binding site. However, direct evidence for the involvement of serine403 has not been reported. In this study, a method which combines liquid chromatography/electrospray mass spectrometry (LC/MS) and nano-electrospray MS for the identification of the active site serine in PBP2a is described. The covalent binding of the beta-lactams was carried out in vitro with the recombinant PBP2a. Peptide mapping of the cyanogen bromide fragments from penicilloyl-PBP2a, using microbore LC/MS, provided a rapid identification of the modified peptide with a 334 Da mass increase. The acylated peptide was isolated and further digested with trypsin. Nano-electrospray MS/MS sequencing of the acylated peptide in the tryptic digest showed that the penicillin was indeed attached to serine403.