SOD activity of immobilized enzyme mimicking complexes

A binuclear, imidazolato-bridged, possible superoxide dismutase-mimicking complex (Cu(II)-diethylenetriamino-μ-imidazolato-Zn(II)-tris-aminoethylamine-triperchlorate) was prepared and immobilized on silica gel or among the layers of montmorillonite. The superoxide dismutase (SOD) activity of the complex before and after immobilization was studied by a SOD assay. It was found that the SOD activity of the host-free complex decreased somewhat when montmorillonite was the host, however, using silica gel as host it increased

Cloning, preparation and preliminary crystallographic studies of penicillin V acylase autoproteolytic processing mutants

The crystallization of three catalytically inactive mutants of penicillin Vacylase (PVA) from Bacillus sphaericus in precursor and processed forms is reported. The mutant proteins crystallize in different primitive monoclinic space groups that are distinct from the crystal forms for the native enzyme. Directed mutants and clone constructs were designed to study the post-translational autoproteolytic processing of PVA. The catalytically inactive mutants will provide threedimensional structures of precursor PVA forms, plus open a route to the study of enzyme-substrate complexes for this industrially important enzyme

The synthesis and the catalytic (catalase and tyrosinase) activities of amino acid copper complexes covalently grafted onto silica gel

In this work the synthesis, structure and certain catalytic properties of amino acid copper complexes covalently grafted onto silica gel are described. The following enzyme mimicking complexes were synthesized and characterised by experimental (FT-IR) and computational (mainly MM+) methods: BOC-His-Cu/silica gel, BOC-Tyr-Cu/silica gel, His-OMe-Cu/silica gel, Tyr-OMe-Cu/silica gel, H-His-Cu/silica gel, H-Tyr-Cu/silica gel His-OH-Cu/silica gel and Tyr-OH-Cu/silica gel. The activities of these substances were also tested in the decomposition of hydrogen peroxide. The majority of the substances proved to be good enzyme mimics displaying either catalase or tyrosinase activity

Covalently bound substrate at the regulatory site triggers allosteric enzyme activation

The mechanism by which the enzyme pyruvate decarboxylase from yeast is activated allosterically has been elucidated. A total of seven three-dimensional structures of the enzyme, of enzyme variants or of enzyme complexes form two yeast species (three of them reported here for the first time) provide detailed atomic resolution snapshots along the activation coordinate. The prime event is the covalent binding of the substrate pyruvate to the side chain of cysteine 221, thus forming a thiohemiketal. This reaction causes the shift of a neighbouring amino acid, which eventually leads to the rigidification of two otherwise flexible loops, where one of the loops provides two histidine residues necessary to complete the enzymatically competent active site architecture. The structural data are complemented and supported by kinetic investigations and binding studies and provide a consistent picture of the structural changes, which occur upon enzyme activation

Bioinspired catalysts: Synthesis, characterisation and some applications

Our recent work concerning the synthesis, characterisation and some applications of bioinspired electron-transfer catalysts is reviewed in this contribution. The catalysts were various mono- or heterobimetallic complexes having either Cu(II) or Cu(II) and Zn(II) as central ions and amino acids, their derivatives or various N-containing organic molecules as ligands. Emphasis was based upon the solid support immobilised versions of these complexes. They were built or anchored onto various kinds of supports (silica gel, montmorillonite, Merrifield’s resin) with different methods (adsorption, ion exchange, covalent grafting). The resulting materials were characterised by a variety of instrumental (FT-IR, Raman, EPR [electron paramagnetic resonance] and atomic absorption spectroscopies, thermogravimetry) as well as computational methods. Their superoxide dismutase, catecholase and catalase activities were tested and some of them were found to be promising candidates as durable electron-transfer catalysts being close to the efficiency of the mimicking enzymes

Snf2 family ATPases and DExx box helicases:differences and unifying concepts from high-resolution crystal structures

Proteins with sequence similarity to the yeast Snf2 protein form a large family of ATPases that act to alter the structure of a diverse range of DNA–protein structures including chromatin. Snf2 family enzymes are related in sequence to DExx box helicases, yet they do not possess helicase activity. Recent biochemical and structural studies suggest that the mechanism by which these enzymes act involves ATP-dependent translocation on DNA. Crystal structures suggest that these enzymes travel along the minor groove, a process that can generate the torque or energy in remodelling processes. We review the recent structural and biochemical findings which suggest a common mechanistic basis underlies the action of many of both Snf2 family and DExx box helicases

A robust and efficient method for estimating enzyme complex abundance and metabolic flux from expression data

A major theme in constraint-based modeling is unifying experimental data, such as biochemical information about the reactions that can occur in a system or the composition and localization of enzyme complexes, with highthroughput data including expression data, metabolomics, or DNA sequencing. The desired result is to increase predictive capability resulting in improved understanding of metabolism. The approach typically employed when only gene (or protein) intensities are available is the creation of tissue-specific models, which reduces the available reactions in an organism model, and does not provide an objective function for the estimation of fluxes, which is an important limitation in many modeling applications. We develop a method, flux assignment with LAD (least absolute deviation) convex objectives and normalization (FALCON), that employs metabolic network reconstructions along with expression data to estimate fluxes. In order to use such a method, accurate measures of enzyme complex abundance are needed, so we first present a new algorithm that addresses quantification of complex abundance. Our extensions to prior techniques include the capability to work with large models and significantly improved run-time performance even for smaller models, an improved analysis of enzyme complex formation logic, the ability to handle very large enzyme complex rules that may incorporate multiple isoforms, and depending on the model constraints, either maintained or significantly improved correlation with experimentally measured fluxes. FALCON has been implemented in MATLAB and ATS, and can be downloaded from: https://github.com/bbarker/FALCON. ATS is not required to compile the software, as intermediate C source code is available, and binaries are provided for Linux x86-64 systems. FALCON requires use of the COBRA Toolbox, also implemented in MATLAB.Comment: 30 pages, 12 figures, 4 table