Recovery Of Catalytic Activity Research Articles

Reaction of trichloroethylene oxide with proteins and dna: instability of adducts and modulation of functions.

Trichloroethylene (TCE) shows several types of toxicities, some of which may be the result of bioactivation. Oxidation by P450s yields the electrophile TCE oxide. We previously analyzed N(6)-acyllysine adducts formed from the reaction of TCE oxide with proteins [Cai, H., and Guengerich, F. P. (2000) Chem. Res. Toxicol. 13, 327-335]; however, we had been unable to measure ester adducts under the prolonged conditions of proteolysis and derivatization. Protein amino acid adducts were directly observed by mass spectrometry during the reaction of TCE oxide with the model polypeptides insulin and adrenocorticotropic hormone (ACTH, residues 1-24). The majority (80%) of the protein adducts were unstable under physiological conditions and had a collective t(1/2) of approximately 1 h, suggesting that they are ester type adducts formed from reactions of Cys, Ser, Tyr, or Thr residues with intermediates formed in TCE oxide hydrolysis. Synthetic O-acetyl-L-Ser and O-acetyl-L-Tyr had half-lives of 1 h and 10 min at pH 8.0, respectively, similar to the stabilities of the protein adducts. The effects of TCE oxide adduct formation on catalytic activities were examined with five model enzymes. No recovery of catalytic activity was observed during the reaction of TCE oxide with two model enzymes for which the literature suggests roles of a Lys, rabbit muscle aldolase and glucose-6-phosphate dehydrogenase. However, in the cases of papain (essential Cys residue in the active site), alpha-chymotrypsin (critical Ser residue), and D-amino acid oxidase (essential Cys and Tyr residues), time-dependent recoveries of enzyme activity were observed following reaction with TCE oxide or either of two model nucleophiles (dichloroacetyl chloride and acetic formic anhydride), paralleling the kinetics of removal of adducts from insulin and ACTH. Formation of adducts ( approximately 2%) was detected in the direct reaction of TCE oxide with 2'-deoxyguanosine, but not with the other three nucleosides found in DNA. During the reaction of TCE oxide with a synthetic 8-mer oligonucleotide, formation of adducts was observed by mass spectrometry. However, the adducts had a t(1/2) of 30 min at pH 8.5. These results indicate the transient nature of the adducts formed from the reaction of TCE oxide with macromolecules and their biological effects.

Recovery of Catalytic Activity from an Inactive Aggregated Mutant of [formula omitted]-Aspartase

Two highly conserved lysyl residues have been replaced with an arginine to examine their role in the mechanism of l-aspartase from Escherichia coli. Replacement of an active-site lysine results in a significant loss of catalytic efficiency [A. S. Saribas, J. F. Schindler, and R. E. Viola (1994) J. Biol. Chem. 269, 6313–6319], while replacement of the second lysine leads to a completely inactive and insoluble protein. Fluorescence spectral evidence has suggested that the loss of activity is due to the misfolding of this aspartase mutant. Some catalytic activity is recovered when the mutant is treated with varying levels of denaturants, and extended treatment with high levels of guanidine.HCl results in the recovery of a substantial fraction of the wild-type activity from this inactive mutant. However, upon removal of the denaturant this mutant enzyme slowly reverts to its inactive and insoluble form. Treatment with an artificial chaperone system in which solubilization by detergent is followed by its removal with β-cyclodextrin leads to a stable enzyme under nondenaturing conditions with about half the catalytic activity of the wild-type enzyme. These results confirm a structural role for lysine-55 in l-aspartase and demonstrate that additional characterization is required before conclusions can be drawn from the production of an inactive mutant.

Refolding of urea-denatured adenylate kinase.

The refolding of urea-denatured adenylate kinase (EC 2.7.4.3) has been followed by formation of the secondary structure, change of surface hydrophobicity and recovery of catalytic activity. During refolding of adenylate kinase with a 20-80-fold dilution of 4 M urea-denatured enzyme at 10 degrees C, the formation of the secondary structure is a fast process with a rate constant of >0.16 s-1. Transient enhancement of the 8-anilino-1-naphthalenesulphonate (ANS) fluorescence intensity is followed by a fluorescence decrease to the level equal to the value characteristic of native enzyme. The desorption of ANS binding fluorescence is relatively slow and can be fitted to a first order reaction with a rate constant of 0.004 s-1 when the ANS is present in the dilution buffer. The desorption of ANS-binding fluorescence is accelerated in the presence of nucleotide substrates. The rate constants are increased to 0.049, 0. 029, 0.028 and 0.029 s-1 in the presence of 1 mM AMP, MgATP, ATP and ADP respectively. The refolding rate constant calculated from the initial fluorescence intensity after mixing ANS with protein at different refolding intervals is 0.016 s-1, which is faster than those obtained when ANS is present throughout the refolding process, indicating that the binding of ANS with a partially folded intermediate retards its further refolding to its native structure. The reactivation rate is even faster than the rates of refolding monitored in the absence of substrates, showing that the refolding is accelerated in the presence of the substrates. A possible refolding pathway and the accelerating effect of substrates are discussed.

Reversible Acylation of Factor Xa as a Potential Therapy for Hemophilia

Current therapies for treatment of hemophilia A involve infusion of factor VIII, but are ineffective for patients who develop inhibitory antibodies. We have previously proposed that bypassing the intrinsic pathway (VIIIa/IXa) with reversibly acylated factor Xa offers an improvement on existing therapies as it provides a time-dependent release of procoagulant activity without the addition of factors VIII or IX. The present study was designed to determine the effect of substituted 4-amidinophenyl benzoates on the acylation of factor Xa, as well as the subsequent deacylation rates of the resulting acyl Xa. A subset of this series of acyl Xa's were incorporated into the prothrombinase complex and recovery of catalytic activity was measured by activation of prothrombin to thrombin. Similarly, some acyl Xa's were also evaluated for their capacity to enhance clotting times of human plasma. Our study indicates that by choosing the appropriate acyl Xa, the time course of factor Xa regeneration can be modulated extensively. Animal studies will be required to show that the use of acyl Xa as a procoagulant agent is feasible in an in vivo system.

Transferred nuclear Overhauser effect study of the C-terminal helix of yeast phosphoglycerate kinase: NMR solution structure of the C-terminal bound peptide.

Two-dimensional 1H nuclear magnetic resonance spectroscopy is used to determine the structure of the C-terminal complementary peptide (404-415) bound to a mutant phosphoglycerate kinase (1-403). Conformational changes in the peptide induced by the formation of the peptide-protein complex are followed by transferred nuclear Overhauser effect spectroscopy. Measurement of transferred NOEs and molecular modeling reveal an alpha-helix fold in the 405-409 region. This fold is in good agreement with the corresponding helix XIV of the crystallographic structure of wild-type PGK (Watson et al., 1982). The role of the alpha-helix from the C-terminal peptide in the recovery of catalytic activity in the mutant PGK is discussed.

Unfolding of 4-aminobutyrate aminotransferase equilibrium and kinetic studies

The unfolding of pig liver 4-aminobutyrate aminotransferase by urea has been investigated at equilibrium. The overall process was reversible as judged from the recovery of catalytic activity after dilution of urea-treated samples. Unfolding of the enzyme was monitored by circular dichroism and fluorescence spectroscopy. The steepness of the fluorescence and CD changes between 2 and 8 M urea, and the lack of any discernible plateau suggests that unfolding of the protein is a cooperative process. The unfolding of 4-aminobutyrate aminotransferase as a function of urea concentration was monitored by fluorescence measurements of the tryptophanyl residues. The kinetic results indicate that the aminotransferase unfolds in a single kinetic phase. Unfolded 4-aminobutyrate aminotransferase recovers immediately its catalytic activity upon dilution with buffers of neutral pH. Based on equilibrium and kinetic results, a two- state model for the unfolding of the aminotransferase is proposed.

Interaction of 70-kDA heat shock cognate protein with peptides and myo-inositol monophosphatase.

Fluorescence techniques have been used to investigate the interaction of bovine 70-kDa heat shock cognate protein (Hsc 70) with small molecular weight peptides and myo-inositol monophosphatase. The emission properties of Hsc 70 remain invariant upon addition of ATP. The results of steady-state fluorescence indicate that the tryptophan residues of Hsc 70 are exposed to the rapidly relaxing aqueous solvent. Binding of residues 1-20 of ribonuclease A (RNase S-peptide) to Hsc 70 causes protein fluorescence quenching which was used to determine a dissociation constant Kd = 2.7 microM for the binary Hsc 70.RNase S-peptide complex. The octapeptide corresponding to the NH2-terminal portion of sickle cell hemoglobin recognizes Hsc 70 and binds with a Kd = 9.3 microM. Binding of RNase S-peptide to Hsc 70 produces a small enhancement of ATPase activity. Unfolded myo-inositol monophosphatase, tagged with the fluorescent probe 5-[2-(2-iodoacetamido)ethylamino]-1-naphthalenesulfonic acid, recognizes Hsc 70; the formation of a stable complex was detected by steady-state emission anisotropy measurements. The rate and extent of recovery of catalytic activity of unfolded myo-inositol monophosphatase is not influenced by Hsc 70. It is suggested that interaction of Hsc 70 with unfolded proteins in the cell may be able to delay the formation of misfolded structures.

Reversible unfolding of pyridoxal kinase.

The unfolding of brain pyridoxal kinase by guanidinium HCl has been investigated at equilibrium. The overall process was reversible as judged from the complete recovery of catalytic activity after removal of guanidinium HCl. Unfolding of pyridoxal kinase was monitored by circular dichroism and fluorescence spectroscopy. The steepness of the spectroscopic changes between 0.2 and 1.5 M guanidinium HCl, and the lack of any discernible plateau suggests that unfolding of the monomer is a cooperative process. A compact intermediate on the unfolding pathway of pyridoxal kinase could not be detected by the method of denaturant gel filtration. The fluorescent analogs of the substrates ATP and pyridoxal were used to assess differences in stability among the domains of the protein. Based on fluorescence and steady emission anisotropy results, it is postulated that the nucleotide domain is more stable than the pyridoxal domain of the kinase.

A basic study of catalyst aging in the H-coal process

Samples of CoMo/Al 2O 3 catalysts used in an H-coal process demonstration run were studied to determine causes of catalyst deactivation. Physical and surface properties of the aged and regenerated catalysts were examined. Model compounds were used to assess four catalyst activity functions, viz., hydrodesulfurization (HDS), hydrogenation, cracking and hydrodeoxygenation (HDO). Other tests were performed to study the effects of coke and metals separately on the four catalyst activity functions. Catalyst coke content and metals deposits first increased rapidly, then more gradually with exposure time in the process run. Surface area and pure volume markedly decreased with exposure time. Catalyst activities of aged catalysts showed a rapid decline with exposure time. One-day exposure to coal resulted in significant losses in HDS and hydrogenation activities and nearly complete loss in cracking and HDO activities. Although metal deposits caused some permanent catalyst deactivation, coke had a much greater effect. Regenerated catalysts showed less recovery of catalytic activity as processing time increased. These results agreed well with product inspections from the process run. Oxygen chemisorption on aged—regenerated catalysts decreased with catalyst exposure time, indicating a significant loss of active sites. However, ESCA results showed no evidence of extensive sintering of the active MoS 2 phase. Permanent deactivation of the longer-time exposed catalysts can be ascribed, at least partly, to lateral growth of the active molybdenum sulfide phase. In addition, some loss in cobalt promotion occurred early in the process, which may account for the rapid loss in HDS and HDO activity in regenerated catalysts.

Purification of F 1-ATPase with impaired catalytic activity from partial revertants of Escherichia coliuncA mutant strains

It is shown that F 1-ATPase preparations having impaired catalytic rates may be purified from partial revertants of uncA mutant strains of Escherichia coli. Recovery of catalytic activity in the partial revertant F 1 was accompanied by recovery of α ↔ β intersubunit conformational interaction, supporting the hypothesis that such interaction is required for normal catalysis in F 1. The specific ATPase activities of the partial revertant F 1 preparations were in the range 1–29% of normal, and some of the preparations showed unusual insensitivity to inhibitors. The properties of a new uncA mutant F 1 preparation ( uncA498) which has approximately half of normal catalytic rate are also briefly described.

Immobilized lipoxygenase in continuous production of fatty acid hydroperoxides

AbstractSoybean lipoxygenase‐1 was covalently coupled to agarose with 75% recovery of catalytic activity. Because evidence was obtained that the immobilization resulted in improved operational stability of the enzyme, a lipoxygenase‐reactor and a continuous process for the synthesis of 13‐hydroperoxy‐linoleic acid and 15‐hydroperoxyarachidonic acid were developed. A procedure based on spectrophotometric hydroperoxide assay and constant oxygraphic monitoring of the effluent is presented for the calibration of the reactor to operate at the highest conversion efficiency when oxygenating quantitatively the substrate. Under these conditions, the reactor was capable of producing about 0.6 mg of hydroperoxy fatty acid/1.0 ml of wet gel/hr. The covalently coupled enzyme has been stable during six months of storage at 3 C in 0.2 M Na‐borate buffer, pH 9.0, and during the same period, its operational stability in the column has been unaltered under the conditions used.

Purification, stabilization, and characterization of rat hepatic triglyceride lipase

Hepatic triglyceride lipase (H-TGL) was purified to near homogeneity from heparin-containing rat liver perfusates with the following column chromatography steps: heparin-Sepharose affinity chromatography, anion-exchange chromatography on DEAE-Sephacel, and gel filtration on Ultrogel AcA 34. A final specific activity of 45,000 μmol fatty acid/mg/h was obtained with an overall 31% recovery of catalytic activity. The heparin-Sepharose step resulted in a 20-fold purification, while the DEAE and gel filtration steps led to further purification with complete recovery of activity. An extensive survey of various detergents as potential stabilizers of H-TGL activity led to the selection of Triton N-101 for use in the column buffers of the DEAE and gel filtration steps. Relative to initial H-TGL activity upon dilution in buffer without detergent, recoveries between 90 and 100% were consistently obtained with Triton N-101-containing buffers following a 24-h incubation at 20°C. In contrast after a 24-h incubation at 20°C those control samples lacking detergent were at least 95% inactivated. The highly purified H-TGL exhibited a single major band by sodium dodecyl sulfate-electrophoresis. The use of DEAE chromatography and stabilization of H-TGL with Triton N-101 are the improvements in purification that resulted in an 8-fold enhancement in specific activity relative to the highest previous report of purification from rat liver perfusates.

The Separation and Properties of Two Penicillin-Binding Proteins from Salmonella typhimurium

The cell envelope of Salmonella typhimurium SW 1061 contains five proteins which covalently bind benzyl-[14C]penicillin, numbered 1–5 in order of decreasing molecular weight. Proteins 1. 4 and 5 were extracted from membranes of the organism by treatment with Genapol X-100 in the presence of 1 M LiCl. This extract catalysed a dd-carboxypeptidase reaction, the synthesis of a cross-linked dimer from l-Ala-d-Glu-(ms-[1,7−14C]A2pm-d-Ala) in a natural model transpeptidation reaction, and the concomitant hydrolysis of this dimer, i.e. an endopeptidase reaction. Proteins 1. 4 and 5 have been separated by ion-exchange chromatography of the LiCl/Genapol X-100 extract on DEAE-Sepharose CL-6B, and protein 4 was found to possess the majority of the recovered dd-carboxypeptidase activity, and to be an efficient transpeptidase. However, this procedure yielded unstable preparations of protein 5 (the major penicillin-binding protein in the cell), which had very low enzymic activity but retained the ability to bind benzyl-[14C]penicillin. Stable, pure preparations of protein 5 were obtained by covalent affinity chromatography on ampicillin-affinose of material solubilized from membranes using Genapol X-100 in the absence of LiCl. Such preparations possessed dd-carboxypeptidase activity, and also catalysed the natural model transpeptidation reaction but with a lower efficiency than protein 4. Proteins 4 and 5 released benzylpenicillin with a concomitant recovery of catalytic activity, the half-times for this recovery being approximately 90 min and 5 min respectively at 37 °C. Protein 4 was found to have the highest affinity of the five membrane-bound penicillin-binding proteins for benzyl-[14C]penicillin. The complete separation of protein 1 from proteins 4 and 5 has not been achieved; however, our preliminary findings suggest that this protein also catalyses both the dd-carboxypeptidase and natural model transpeptidase activities.

Mechanism of action of Zn2+ and Mg2+ on rat placenta alkaline phosphatase. II. Studies on membrane-bound phosphatase in tissue sections and in whole placenta.

Alkaline phosphatase (EC 3.1.3.1) bound to trophoblastic cells in rat placenta is activated by Mg2+ and inhibited by Zn2+ in the same way as is found with partially purified soluble alkaline phosphatase in the same tissue (PetitClerc, C., Delisle, M., Martel, M., Fecteau, C. & Brière, N. (1975) Can. J. Biochem. 53, 1089-1100). In studies done with tissue sections (6-10 micron), it is shown that alkaline phosphatase activity and labelling of active sites by orthophosphate are lost during incubation with ethanolamine at pH 9.0. Addition of Mg2+ causes total recovery of catalytic activity and active sites labelling. Zn2+ displaces and replaces at the Mg2+ binding sites. The affinity for both ions is similar, and dissociation of Zn2+ from the enzyme is a very slow process, even in the presence of Mg2+. The Zn2+-alkaline phosphatase and Mg2+-alkaline phosphatase, which only differ by the ion bound to an apparent modulator site, have the same catalytic activity at pH less than 7.0, but the Zn2+ species has little activity at alkaline pH. Phosphorylation of the enzyme by orthophosphate indicates that with both enzyme species phosphoryl intermediate does not accumulate at alkaline pH. These results suggest that with orthophosphate, the phosphorylation step is rate determining for both enzymes, and that Zn2+ affects this step to a much greater extent. It is proposed that Zn2+ and Mg2+ regulate alkaline phosphatase in rat placenta. The concentration of both ions in maternal serum and placenta suggest that such a mechanism could exist in vivo.

Role of Oxalacetate in the Regulation of Mammalian Succinate Dehydrogenase

Abstract In most procedures for the isolation of membrane-bound or soluble succinate dehydrogenase, the enzyme is obtained largely in the deactivated form. As judged by the quantity of oxalacetate enzymatically determined in perchlorate extracts, the deactivated form contains tightly bound oxalacetate in 1:1 proportion to the enzyme (equated with histidyl flavin content) in particles. A somewhat lower ratio has been obtained for soluble preparations of the enzyme. The oxalacetate is bound very tightly: it is not displaced by succinate or malonate in the cold, nor released by precipitation or gel exclusion of the enzyme, and it does not react with malate dehydrogenase. It is released, however, on denaturation of the enzyme with perchloric acid or under conditions which favor conversion of the enzyme to the activated form. If the enzyme is freed from oxalacetate by activation with bromide and gel exclusion, treatment of the oxalacetate-free fully active enzyme with very low concentrations of l- or d-malate or oxalacetate leads to deactivation and concomitant formation of tightly bound oxalacetate in the same molar ratio found in the enzyme as isolated from heart mitochondria. Dissociation of the complex formed in this manner requires the same experimental conditions as those needed for dissociation of the complex as found in the isolated enzyme. The activation energies for activation and for release of oxalacetate are the same but vary with the type of activator used. The first order rate constant for activation by a variety of agents is about twice that measured for the dissociation of tightly bound oxalacetate. Moreover, with some activators oxalacetate release shows a considerable lag behind recovery of catalytic activity. It is suggested that the presence of oxalacetate in the deactivated enzyme is not the cause of its lack of catalytic activity but a consequence of the deactivated conformation. Further observations are described which suggest that a deactivation-activation cycle may also occur in the absence of oxalacetate.