Publisher Summary This chapter investigates the anatomy and taxonomy of protein structures. A protein is a polypeptide chain made up of amino acid residues linked together in a definite sequence. Amino acids are “handed,” and naturally occurring proteins contain only L-amino acids. A simple mnemonic for that purpose is the “corncrib.” The sequence of side chains determines all that is unique about a particular protein, including its biological function and its specific three-dimensional structure. The major possible routes to knowledge of three-dimensional protein structure are prediction from the amino acid sequence and analysis of spectroscopic measurements such as circular dichroism, laser Raman spectroscopy, and nuclear magnetic resonance. The analysis and discussion of protein structure is based on the results of three-dimensional X-ray crystallography of globular proteins. The basic elements of protein structures are discussed. The most useful level at which protein structures are to be categorized is the domain, as there are many cases of multiple-domain proteins in which each separate domain resembles other entire smaller proteins. The simplest type of stable protein structure consists of polypeptide backbone wrapped more or less uniformly around the outside of a single hydrophobic core. The outline of the taxonomy is also provided in the chapter.

The anatomy and taxonomy of protein structure.

Publisher Summary This chapter presents a procedure for the preparation of glutathione peroxidase, which is regarded as a major protective system against endogenously and exogenously induced lipid peroxidation. Two types of methods are used for determining the activity of glutathione peroxidase. One involves a direct measurement of unconsumed glutathione (GSH) at fixed time periods by polarographic GSH analysis' (Method 1), or by the dithionitrobenzoic acid method (Method 2). The second approach takes advantage of the capability of glutathione reductase, with nicotinamide adenine dinucleotide phosphate (NADPH), to regenerate GSH from oxidized GSH. The decrease in NADPH is continuously measured spectrophotometrically, while the GSH concentration in the enzymatic cycle remains essentially constant (Method 3). A convenient source for the preparation of glutathione peroxidase is bovine blood including the following steps: hemolysate; organic solvent precipitation; phosphate precipitation; absorption to phenyl-sepharose; and washing on diethylaminoethyl (DEAE)–sephadex, S-300 sephacryl, and hydroxylapatite column.

[44] Glutathione peroxidase

Reactive oxygen species, antioxidants, and the mammalian thioredoxin system.

Glutathione metabolism and its selective modification.

Mice deficient for the 55 kd tumor necrosis factor receptor are resistant to endotoxic shock, yet succumb to L. monocytogenes infection.

The crystal structure of bovine erythrocyte glutathione peroxidase has been refined by a combined procedure of restrained crystallographic refinement and energy minimization at 0.20 nm resolution. The final R value at this resolution is 0.178. The r.m.s. deviation of main-chain atoms of the two independently refined monomers is 0.019 nm. The structure at 0.28 nm resolution, which has been determined by multiple isomorphous replacement, served as a starting model.

The refined model allowed a detailed survey of the hydrogen-bonding pattern and of the subunit contact areas in the molecule. The model contains 165 solvent molecules per dimer, all taken as water molecules. The mobility of the structure was derived from the individual atomic temperature factors. The complete tetramer, including the active sites, seems to be rather rigid, except for narrow loops near to the N-terminal ends and some β turns exposed to solvent.

The active centres of glutathione peroxidase are found in flat depressions on the molecular surface. The catalytically active selenocysteine residues could be located at the N-terminal ends of α helices forming βαβ substructures together with two adjacent parallel β strands. In the vicinity of the reactive group some aromatic amino acid side-chains could be localized. Especially Trp-148, which could be hydrogen bonded to SeCys-35, may play a functional role during catalysis.

The results of substrate and inhibitor binding studies in solution and in the crystalline state could be interpreted by an apparent half-site reactivity of glutathione peroxidase. The enzyme seems to react in the sense of negative cooperativity with dimers being the functional units.

Based on difference Fourier analyses of appropriate derivatives a reasonable model of glutathione binding is presented. Among the residues which could be of functional importance are Arg-40, Gln-130 and Arg-167, presumably forming salt bridges and a hydrogen bond to the glutathione molecule.

In conclusion, a general picture of a minimal reaction mechanism, which is in good agreement with functional and structural data, is proposed. The main reaction of the catalytic cycle presumably shuttles between the selenolate and the selenenic acid state of SeCys-35.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1983.tb07429.x

The refined structure of the selenoenzyme glutathione peroxidase at 0.2-nm resolution.

Acute paracetamol intoxication of starved mice leads to lipid peroxidation in vivo

The fate of extracellular glutathione in the rat.

Drug-induced lipid peroxidation in mice--I. Modulation by monooxygenase activity, glutathione and selenium status.

Selenium-containing glutathione peroxidase (EC 1.11.1.9) was purified 6000-fold from bovine red blood cells to apparent homogeneity. Lipoxygenase (EC 1.13.11.12) was enriched 20-fold from soybean acetone powder. Linoleic acid was peroxidized with lipoxygenase and then used as a substrate in the glutathione peroxidase reaction. Analogous experiments were conducted with synthetic 1,2-dilinoleoyl-l-α-glycerophosphocholine and with natural bovine heart cardiolipin. The peroxidized phospholipids were reactive with glutathione peroxidase only after enzymatic attack by phospholipase A2 (EC 3.1.1.4). This result implies that the membrane-protective function of glutathione peroxidase includes preceeding phospholipase action and excludes a direct interaction of this enzyme with membrane-bound lipid hydroperoxides.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1983.tb07687.x

Albrecht Wendel

Papers

The refined structure of the selenoenzyme glutathione peroxidase at 0.2-nm resolution.

Acute paracetamol intoxication of starved mice leads to lipid peroxidation in vivo

The fate of extracellular glutathione in the rat.

Drug-induced lipid peroxidation in mice--I. Modulation by monooxygenase activity, glutathione and selenium status.

Non-reactivity of the selenoenzyme glutathione peroxidase with enzymatically hydroperoxidized phospholipids