Membrane-integrated enzymes

The most complex monooxygenases, however, are the membrane integrated enzyme complexes occurring in liver and adrenocortex of mammals, which are involved in steroid hydroxylation (D 6.4.5). These complexes possess special... 

He/minthosporium (15). The mode of action is considered to be inhibition of the enzyme NADPH-cytochrome C reductase, which results in the generation of free radicals and/or peroxide derivatives of flavin which oxidize adjacent unsaturated fatty acids to dismpt membrane integrity (16) (see Enzyme inhibitors). 

Complex II is perhaps better known by its other name—succinate dehydrogenase, the only TCA cycle enzyme that is an integral membrane protein in the inner mitochondrial membrane. This enzyme has a mass of approximately 100 to 140 kD and is composed of four subunits two Fe-S proteins of masses 70 kD and 27 kD, and two other peptides of masses 15 kD and 13 kD. Also known as flavoprotein 2 (FP2), it contains an FAD covalently bound to a histidine residue (see Figure 20.15), and three Fe-S centers a 4Fe-4S cluster, a 3Fe-4S cluster, and a 2Fe-2S cluster. When succinate is converted to fumarate in the TCA cycle, concomitant reduction of bound FAD to FADHg occurs in succinate dehydrogenase. This FADHg transfers its electrons immediately to Fe-S centers, which pass them on to UQ. Electron flow from succinate to UQ,... 

In both plant (e.g. [57]) and animal (e.g. [86]) cell systems, cellular respiration has been shown to be a more sensitive indicator of system response to hydro-dynamic stress than membrane integrity, suggesting that intracellular enzymes and/or organelles may be affected at stress levels lower than those required to cause membrane damage. 

Membrane-integrated proteins were always hard to express in cell-based systems in sufficient quantity for structural analysis. In cell-free systems, they can be produced on a milligrams per milliliter scale, which, combined with labeling with stable isotopes, is also very amenable forNMR spectroscopy [157-161]. Possible applications of in vitro expression systems also include incorporation of selenomethionine (Se-Met) into proteins for multiwavelength anomalous diffraction phasing of protein crystal structures [162], Se-Met-containing proteins are usually toxic for cellular systems [163]. Consequently, rational design of more efficient biocatalysts is facilitated by quick access to structural information about the enzyme. 

In our previous research, we found that the antialgal allelochemical Ethyl 2-Methylacetoacetate (EMA) caused loss of cell membrane integrity. It hinted that EMA may cause a change in the membrane. It is reported that environmental stress may increase the concentration of ROS in plant cell. The excessive ROS may cause a decrease of antioxidation enzymes activity and lipid peroxidation. The effect of EMA on the activity of SOD and POD and lipid fatty acids of Chlorella pyrenoidosa, Chlorella vulagaris and Microcystis aeruginosa were evaluated to elucidate the mode of action of EMA. 

Figure 2. Behavior of membrane-associated lipases. From left to right (a) catalytic action of an enzyme that first requires attachment to the substrate at the water-membrane interface (b) action of an integral membrane enzyme that remains attached to the membrane where the enzyme finds its substrate (c) action of a membrane-bound enzyme on substrates in the aqueous medium and (d) action of an enzyme in the aqueous phase on a substrate that must first desorb from the membrane before it can interact with enzyme. From Jain et al. with permission of the authors.

Surveying our present knowledge about the enzyme activities in PVA biodegradation, a trend toward increasing integration can be seen. There are free enzymes working in the extracellular space of the cells, including also the periplasmatic volume, and there are membrane-associated enzymes that are presumably Unked to the cellular cytochrome-based electron transport chains. 

Previous studies have shown that muscle lysosomal hydrolases are released early in the postmortem period due to a decrease in intracellular ATP concentrations. The decreased intracellular ATP level causes the rupture of the lysosomal membrane (14), releasing hydrolytic enzymes (proteases, lipases, and glycosidases) that further potentiate the weakening of membrane integrity and cellular function. Furthermore, as the acidosis increases (due to the anaerobic conditions associated with cellular death) the intramuscular pH to levels reach that which are optimal for the activity of several lysosomal thiol proteinases. 

As with other multisubunit enzymes (e.g., allosteric enzymes), the structural integrity of a membrane-bound enzyme primarily is maintained by noncovalent interactions such as hydrogen bonding, electrostatics, and hydrophobic interactions. Hydrophobic polypeptides (or hydrophobic portions of polypeptides) apparently are used to anchor the enzymes to the membrane through interactions with phospholipids. Therefore, I would characterize the interaction between the enzyme and membrane as chemical in nature rather than as geometric. ... 

In virtually every animal cell type, the concentration of Na+ is lower in the cell than in the surrounding medium, and the concentration of K+ is higher (Fig. 11-36). This imbalance is maintained by a primary active transport system in the plasma membrane. The enzyme Na+K+ ATPase, discovered by Jens Slcou in 1957, couples breakdown of ATP to the simultaneous movement of both Na+ and K+ against their electrochemical gradients. For each molecule of ATP converted to ADP and I , the transporter moves two K+ ions inward and three Na+ ions outward across the plasma membrane. The Na+K+ ATPase is an integral protein with two subunits (Mr -50,000 and -110,000), both of which span the membrane. 

Among these are the well-known E. coli leader peptidase355 356 and other signal peptidases.357 These are integral membrane proteins that cleave N-terminal signal sequences from proteins incorporated into plasma membranes. Another enzyme of this class is the lexA repressor and protease discussed in Chapter 28. 

As an example of an asymmetric membrane integrated protein, the ATP synthetase complex (ATPase from Rhodospirillum Rubrum) was incorporated in liposomes of the polymerizable sulfolipid (22)24). The protein consists of a hydrophobic membrane integrated part (F0) and a water soluble moiety (Ft) carrying the catalytic site of the enzyme. The isolated ATP synthetase complex is almost completely inactive. Activity is substantially increased in the presence of a variety of amphiphiles, such as natural phospholipids and detergents. The presence of a bilayer structure is not a necessary condition for enhanced activity. Using soybean lecithin or diacetylenic sulfolipid (22) the maximal enzymatic activity is obtained at 500 lipid molecules/enzyme molecule. With soybean lecithin, the ATPase activity is increased 8-fold compared to a 5-fold increase in the presence of (22). There is a remarkable difference in ATPase activity depending on the liposome preparation technique (Fig. 41). If ATPase is incorporated in-... 

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