Exploring the therapeutic potential of modern and ancestral phenylalanine/tyrosine ammonia-lyases as supplementary treatment of hereditary tyrosinemia.

Phenylalanine/tyrosine ammonia-lyases (PAL/TALs) have been approved by the FDA for treatment of phenylketonuria and may harbour potential for complementary treatment of hereditary tyrosinemia Type I. Herein, we explore ancestral sequence reconstruction as an enzyme engineering tool to enhance the therapeutic potential of PAL/TALs. We reconstructed putative ancestors from fungi and compared their catalytic activity and stability to two modern fungal PAL/TALs.

Mapping and partial characterization of proteases expressed by a CHO production cell line.

The proteolytic activities expressed by a Chinese hamster ovary (CHO) cell line in the cultivation supernatant during the production of recombinant factor VIII were mapped with a broad spectrum protease assay and a series of different types of protease inhibitors. The destabilizing effect on the product emanated from a metalloproteinase, which could be effectively blocked by chelating agents to lead to product stabilization. Amino acid sequences of the isolated metalloproteinase were found to have sequence homology with matrix metalloproteinases (MMPs) MMP3, MMP10, and MMP12.

EPR investigation of the active site of recombinant human 5-lipoxygenase: inhibition by selenide.

Lipoxygenases are a group of non-heme iron dioxygenases which catalyze the formation of lipid hydroperoxides from unsaturated fatty acids. 5-Lipoxygenase (5LO) is of particular interest for formation of leukotrienes and lipoxins, implicated in inflammatory processes. In this study, electron paramagnetic resonance (EPR) spectroscopy was used to investigate the active site iron of purified recombinant human 5-lipoxygenase (5LO), and to explore the action of selenide on 5LO.

Antioxidant enzymes; possible mechanism of gold compound treatment in rheumatoid arthritis.

Reactive oxygen species play a critical role in inflammatory processes including rheumatoid disorders. Antioxidant therapy strategies have been postulated for the treatment of rheumatoid diseases. In this study, we investigated activities and therapeutic implications of antioxidant enzymes in rheumatoid disorders.

Protein hydration in solution: experimental observation by x-ray and neutron scattering.

The structure of the protein-solvent interface is the subject of controversy in theoretical studies and requires direct experimental characterization. Three proteins with known atomic resolution crystal structure (lysozyme, Escherichia coli thioredoxin reductase, and protein R1 of E. coli ribonucleotide reductase) were investigated in parallel by x-ray and neutron scattering in H2O and D2O solutions.

Effects of buried charged groups on cysteine thiol ionization and reactivity in Escherichia coli thioredoxin: structural and functional characterization of mutants of Asp 26 and Lys 57.

To investigate the role of Asp 26 and Lys 57, two conserved, buried residues, in the redox mechanism of Escherichia coli thioredoxin (Trx), three mutant proteins, Asp 26 --> Ala (D26A), Lys 57 --> Met (K57M), and the double mutant D26A/K57M, were prepared, replacing the charged amino acids with hydrophobic residues with similar sizes. Both the oxidized (Trx-S2) and reduced [Trx-(SH)2] forms of the mutant thioredoxins are fully folded and similar in overall structure to the wild-type protein (wt). The structure of the active site hydrophobic surface is unchanged by the mutation of Asp 26 and Lys 57, since DNA polymerase activity in the 1:1 complex of the T7 gene 5 protein and mutant Trx-(SH)2 shows similar Kd values (approximately 5 nM) for both mutants and wt.

[Study of yeast phosphoglycerate kinase by electron paramagnetic resonance. 1. Modification of protein SH-group by spin labels. Conformational changes of enzyme].

The conditions for modifications of a single SH-group of phosphoglycerate kinase from yeast by maleimid spin label and the mercury-containing spin labels were defined. The time course of immobilization of mercury containing paramagnetic radicals was obtained. Based on changes in EPR spectra we calculated dissociation constants for complexes of phosphoglycerate kinase with 3-phosphoglycerate and sulfate ions.

Selenite incubated with NADPH and mammalian thioredoxin reductase yields selenide, which inhibits lipoxygenase and changes the electron spin resonance spectrum of the active site iron.

Selenite and selenodiglutathione (GS-Se-SG) efficiently inhibited 5-lipoxygenase activity in sonicates of human monoclonal B-lymphocytes. The apparent IC50 of GS-Se-SG was 0.5 microM.

Effect of the tyrosyl radical on the reduction and structure of the Escherichia coli ribonucleotide reductase protein R2 diferric site as probed by EPR on the mixed-valent state.

It was recently shown by EPR that high yields of a sterically constrained mixed-valent species may be formed in radical free protein metR2 of Escherichia coli ribonucleotide reductase by gamma-irradiation at 77 K [Davydov, R., Kuprin, S., Gräslund, A.

Nonideality of water-hexafluoropropanol mixtures as studied by X-ray small angle scattering.

Strong temperature dependent low angle X-ray scattering by trifluoroethanol- and hexafluoro-2-propanol-water mixtures was observed in conditions commonly used in NMR work on peptides. At least two types of molecular effects may contribute to the observed scattering: formation of clathrate hydrate-like aggregates of alcohol with water as have been proposed for the similar system tert-butanol-water (Iwasaki, K. and Fujiyama, T.

NMR studies of binding of 5-FdUDP and dCDP to ribonucleoside-diphosphate reductase from Escherichia coli.

5-Fluoro-2'-deoxyuridine-5'-diphosphate (5-FdUDP) has been synthesised using an original route, previously applied to the synthesis of natural nucleoside diphosphates. The interaction between 5-FdUDP and the enzyme ribonucleoside-diphosphate reductase (EC 1.17.

Conformation of dCDP bound to protein R1 of Escherichia coli ribonucleotide reductase.

Deoxycytidine 5'-diphosphate (dCDP) is a product and competitive inhibitor of ribonucleoside-diphosphate reductase (EC 1.17.4.

Binding of the competitive inhibitor dCDP to ribonucleoside-diphosphate reductase from Escherichia coli studied by 1H NMR. Different properties of the large protein subunit and the holoenzyme.

Ribonucleoside-diphosphate reductase (EC 1.17.4.

Studies on ribonucleoside-diphosphate reductase from Escherichia coli. The product dCDP is a competitive inhibitor and functions as a spectroscopic probe for the substrate binding site; demonstration by enzyme kinetics and 1H NMR.

Ribonucleoside-diphosphate reductase (EC 1.17.4.

[Evaluation of distances in yeast phosphoglycerate kinase molecules using saturation effect ESR spectra at 77 K].

Distances between a spin label attached to the single SH-group of yeast phosphoglycerate kinase and paramagnetic ions bound to the active centre in the complex with ATP were estimated by means of saturation of ESR spectra of the spin label at 77K. The calculations were performed for the ions with g-factor approximately equal to the g-factor of the spin label on IBM PC/AT. Distances between the SH-group of phosphoglycerate kinase and the middle of the active centre of the ATP binding site were estimated by using the stable complex of CrATP.

[Study of the binding of substrates and paramagnetic Mn2+ ions with phosphoglycerate kinase by saturating ESR spectra of a spin-labelled protein].

A single SH-group of phosphoglycerate kinase from yeast was modified by mercury-containing spin label. The saturation curves of ESR spectra of the spin-labeled enzyme were studied. The paramagnetic ions of Mn2+ bound to the centre of ion nonspecific binding or active centre in the complex with ATP can influence the saturation of the spin-labeled enzyme.

PH-jump-induced ADP phosphorylation in mitochondria.

Mitochondria, uncoupled by aging or by freeze-thaw treatment, are able to synthesize ATP from ADP and Pi after a fast increase (but not decrease) in the external pH. The maximal ATP yield (approx. 2.

[Change of constant electric field in man in the course of adaptation to hypokinesia].

A study was carried out of change kinetics of man's constant electric field in the course of adaptation to hypokinesia from normal physical load and vice versa. It has been shown that adaptation, a transitional regime from one physiological state to another, involves similar values of the electric potential difference between the fixed points of human skin.

[Chemical characteristics of conformationally-disequilibrium states of metal-containing proteins].

Properties of conformation-out-equilibrium states of several iron containing proteins, for example, induced by fast changes of active site were investigated. It was shown that structure and chemical properties of out-of-equilbrium states can differ considerably from the equilibrium ones. Spectral characteristics and reactional ability of these molecules change in the way of slow (up to 10(-1)s) conformational relaxation, continuously approaching the equilibrium values.

[Kinetics of dissociation of cyanide peroxidase complex].

The kinetics of dissociation ofHRP--CN- complex after reduction of the enzyme with ditionite was studied. The process goes in two consequent stages with different rate constants. The first stage can be related to the removal of CN- from Fe2+ and formation of out-of-equilibrium state of high-spin enzyme.