Alteration Of Catalytic Properties Research Articles

Effects of Low-Shear Modeled Microgravity on the Characterization of Recombinant β-D-Glucuronidase Expressed in Pichia pastoris

In this study, we used a high-aspect-ratio vessel (HARV), which could model environment of microgravity on ground to investigate for the first time the effects of low-shear modeled microgravity (LSMMG) on the characterization of recombinant β-D-glucuronidase expressed in Pichia pastoris. The β-D-glucuronidase gene (GenBank accession no. EU095019) derived from Penicillium purpurogenum Li-3 encoding β-D-glucuronidase (PGUS) was expressed in P. pastoris GS115 in two different environments of LSMMG and normal gravity (NG). Results manifested that both LSMMG and NG conditions had insignificant effects on temperature and pH activity (optimal temperature and pH were 55 and 5.0°C, respectively) and characteristic stability of recombinant PGUS. However, the catalytic activity of recombinant PGUS expressed under LSMMG was less affected by metal ions and EDTA as compared with that of NG. Furthermore, K (m) value of the recombinant PGUS expressed under LSMMG was nearly one fifth of that under NG (1.72 vs. 7.72), whereas catalytic efficiency (k (cat)/K (m)) of PGUS expressed under LSMMG (13.55) was 3.7 times higher than that of NG (3.61). The results initially reveal the significant alterations in catalytic properties of recombinant enzyme in response to LSMMG environment and have potential application in bioprocessing and biocatalysis.

Expression and catalytic properties of rat liver NADP-isocitrate dehydrogenase under normal conditions, after administration of tumor necrosis factor-α and thioctic acid action

Development of apoptosis is accompanied by a decrease in transcripts of NADP-isocitrate dehydrogenase (NADP-ICDH, EC 1.1.1.42), and also by alterations of catalytic properties from the rat liver enzyme in comparison with control. Administration of thioctic acid increased the level of expression towards normal values. Molecular weight of homogenous preparations of NADP-ICDH purified from livers of control and experimental rats was the same 112 ± 5.8 kDa, however, Km values and pH-optimum changed during induction of apoptosis. Regulation of NADP-ICDH activity by some intermediates of the tricarboxylic acid cycle also differed in these groups.

Expression and catalytic properties of rat liver nadp-isocitrate dehydrogenase under normal conditions, after administration of tumor necrosis factor-α and at thioctic acid action

Development of apoptosis is accompanied by a decrease in transcripts of NADP-isocitrate dehydrogenase (NADP-IDH, E.C. 1.1.1.42.), and also alterations of catalytic properties from the rat liver enzyme in comparison with control. Administration of thioctic acid is increased the level of expression towards normal values. Enzyme preparations NADP-IDH were obtained from rat liver at norm conditions, after introduction of tumor necrosis factor and thioctic acid action under apoptosis. Molecular weight of homogenous preparations of NADP-IDH purified from livers of control and experimental rats was 112±5,8 kDa, however Km values and pH-optimum changed in apoptosis. Regulation of NADP-IDH activity under effect of some intermediates of the tricarboxylic acid cycle also differed in these groups.

An ultrasensitive site-specific DNA recombination assay based on dual-color fluorescence cross-correlation spectroscopy

Site-specific exchange of genetic information is mediated by DNA recombinases, such as FLP or Cre, and has become a valuable tool in modern molecular biology. The so far low number of suitable recombinating enzymes has driven current research activities towards alteration of catalytic properties, such as thermostability or recognition sequences. However, identification and analysis of new mutants requires sensitive in vitro activity assays, which traditionally are based on gel electrophoresis. Here, we describe the development of a new sensitive DNA recombination assay based on dual-color fluorescence cross-correlation spectroscopy (DC-FCCS), which works in homogenous solution and does not require any separation step such as electrophoresis. The assay was validated with unlabeled FLP recombinase and different fluorescently labeled DNA substrates containing the FLP recognition target (FRT). This strategy fulfills all requirements for possible application in high throughput screening and engineering of new site-specific DNA recombinases starting from the FLP-FRT system, and is easily adjustable to other systems like Cre/loxP.

Alteration in Catalytic Properties of Human CYP2D6 Caused by Substitution of Glycine-42 with Arginine, Lysine and Glutamic Acid

The effects of the substitution of glycine at position 42 with various other amino acid residues on the functions of CYP2D6 were studied using debrisoquine (DB) and bunitrolol (BTL) 4-hydroxylations as indices of drug-metabolizing enzymes. The substitution with hydrophobic amino acid residues such as valine and phenylalanine did not affect the enzymatic properties such as reduced CO-difference spectra, microsomal CYP contents and oxidation activities towards DB and BTL. The substitution of glycine-42 with a polar but noncharged amino acid residue (serine) exhibited a similar reduced CO-different spectrum, but the substitution with a charged basic (lysine and arginine) or acidic (glutamic acid) amino acid residue commonly produced a peak at 420 nm in addition to a Soret peak at 450 nm. Cytochrome P450 contents and microsomal contents of G42S, G42K, G42R and G42E estimated spectrophotometrically and estimated by Western blot analysis, respectively, were lower than those of the wild-type. Kinetic analysis revealed that the substitution of glycine-42 with charged amino acid residues such as lysine, arginine and glutamic acid markedly increased the apparent K(m) values for DB and BTL oxidations without remarkable changes in the V(max) values. The subsitution with noncharged amino acid residues such as serine, valine and phenylalanine did not cause such a marked change in the K(m) values. Efficiencies (V(max)/K(m)) as DB and BTL 4-hydroxylases of CYP2D6 mutant proteins having charged amino acid residues were found to be decreased mainly by increasing their K(m) values. These results indicate that the properties of amino acid residues at position 42 affect the behavior of CYP2D6 proteins such as anchoring into ER membranes, conversion of P450 to P420 and incorporation of heme into apoproteins.

Site-directed mutagenesis of the catalytic residues Asp-52 and Glu-35 of chicken egg white lysozyme.

The roles of the catalytic active-site residues aspartic acid-52 and glutamic acid-35 of chicken lysozyme (EC 3.2.1.17) have been investigated by separate in vitro mutagenesis of each residue to its corresponding amide (denoted as D52N and E35Q, respectively). The mutant enzyme D52N exhibits approximately 5% of the wild-type lytic activity against Micrococcus luteus cell walls, while there is no measurable activity associated with E35Q (0.1% +/- 0.1%). The measured dissociation constants for the chitotriose-enzyme complexes were 4.1 microM (D52N) and 13.4 microM (E35Q) vs. 8.6 microM for wild type, indicating that the alterations in catalytic properties may be due in part to binding effects as well as to direct catalytic participation of these residues. The mutant lysozymes have been expressed in and secreted from yeast and obtained at a level of approximately 5 mg per liter of culture by high-salt elution from the cell walls.

Induced appearance of adenylate-deaminating activity in highly purified bovine liver mitochondrial monoamine oxidase

1. 1. Treatment of highly purified preparations of bovine liver mitochondrial monoamine oxidase (monoamine:O 2 oxidoreductase (deaminating), EC 1.4.3.4) with chemical reagents ( e.g. oxidizing agents), which caused reversible qualitative alterations in catalytic properties of monoamine oxidases, induced appearance in the enzyme preparations of an ability to catalyze hydrolytic deamination of AMP (ADP and ATP, contrary to other nucleotides or nucleosides studied, were also deaminated). 2. 2. Enzymatic deamination of AMP by highly purified bovine liver mitochondrial monoamine oxidase, pretreated with oxidized oleic acid, was inhibited by carbonyl reagents (isoniazid, hydroxylamine). These reagents did not inhibit deamination of AMP by purified AMP deaminase from rabbit skeletal muscles. 3. 3. Qualitative alteration in catalytic properties of monoamine oxidase (accompanied by the appearance of AMP-deaminating activity in highly purified monoamine oxidases) probably took place also in vivo in experimental pathological states characterized by accumulation of lipid peroxides in tissues.

Mitochondrial monoamine oxidase of rat liver: Reversible qualitative alterations in catalytic properties

1. 1.|Preincubation of rat liver mitochondrial membranes under aerobic conditions in the presence of Cu 2+ (but not of other metal ions) caused a significant decrease in monoamine oxidase (monoamine: oxygen oxidoreductase, EC 1.4.3.4) activity and induced the ability to deaminate substrates of diamine oxidase (diamine: oxygen oxidoreductase (deaminating), EC 1.4.3.6) (histamine and some diamines) and AMP. At the same time, the content of SH groups in the mitochondrial membranes decreased. 2. 2.|Preincubation of highly purified rat liver mitochondrial monoamine oxidase in the presence of Cu 2+ under aerobic conditions decreased the rate of deamination of monoamines and induced the ability to deaminate histamine, some diamines and AMP. These alterations (transformation) in enzymatic properties of monoamine oxidase were accompanied by a decrease in content of SH groups (from 8 to 1 per 10 5 g of protein) in the enzyme. The decrease in content of SH groups was probably due to their partial oxidation. The modified enzyme was insensitive to specific monoamine oxidase inhibitors (pargyline, tranylcypromine), but it was inhibited by some carbonyl reagents (hydroxylamine, isoniazid). 3. 3.|Qualitative alterations in catalytic properties of monoamine oxidase and the accompanying decrease in content of SH groups were observed after treatment of the enzyme with oxidized oleic acid. 4. 4.|Pretreatment of highly purified rat liver monoamine oxidase with specific irreversible monoamine oxidase inhibitors prevented appearance of the histamine deaminating activity in the course of subsequent incubation in the presence of Cu 2+. 5. 5.|Pretreatment of highly purified monoamine oxidase with alkylating agents ( N-ethylmaleimide, iodoacetamide) did not alter the catalytic properties of monoamine oxidase qualitatively, but prevented the development of these alterations in the course of subsequent incubation of the enzyme in the presence of Cu 2+. 6. 6.|Qualitative alteration in catalytic properties of monoamine oxidase (preincubated in presence of Cu 2+ or treated with oxidized oleic acid) was partially reversed by treatment of the enzyme with reducing agents (NaBH 4, reduced glutathione, Na 3AsO 3).