Dissociable/dissociated enzymes

Fig. 14.21. The dissociated enzyme. (Reprinted from A. Szucs, G. D. Hitchens and J. O M. Bockris, J. Electrochem. Soc. 130 6(12) 3749. Fig. 1,1989. Reproduced by permission of the Electrochemical Society, Inc.)...

However, since the SDS-PAGE mostly dissociates enzyme-inhibitor complexes, the results of zymography fail to give information on the level of the true enzymatic activity of the sample. An example of application of this technique is given below for detection of gelatinolytic activities (Fig. 4.1). 

Current understanding of the mechanism by which calmodulin induces enzyme activation has been discussed in some detail previously (12). All enzymes known to be activated by calmodulin (Table II) with the exception of phosphorylase t> kinase (48), readily dissociate from the protein on anion exchange columns when chromatographed with Ca + chelators. Regulation of dissociable enzymes by calmodulin (C) is generally believed to occur through two sequential, fully reversible mass action expressions ... 

Although the supernatant fraction S-1 [similar to the solubilized preparation described by Sharma et al. (29)] of the mitochondrial sonicate displayed 11 -hydroxylase activity, reaction rates were not proportional to the amount of mitochondrial protein added, but they decreased precipitously with increasing dilution of the preparation (15). This observation, indicating a dissociating enzyme system, prompted the separation of the sample into the particulate fraction and the two soluble components of the reducing system. 

Femandez-Lafuente, R., 2009. Stabihzation of multimeric enzymes strategies to prevent subunit dissociation. Enzyme Microb. Technol. 45 (6—7), 405—418. 

Type II Plants Euglena gracilis (chloroplasts) (a) Dissociable enzymes (6) in a complex with ACP... 

In the present study with maize dikinase, the effect of magnesium on the quaternary structure and the activity of the enzyme were examined. We provide evidence with gel filtration (HPLC) technique that in the absence of magnesium it dissociates to a dimeric form. The dissociated enzymes were reassociated to a tetramer by the addition of magnesium. Absence of magnesium also decreases the activity of the enzyme. The specific activity of the dimeric form seems to be a half of that of the tetramer form. 

Reassociation of the dissociated enzyme after the addition of magnesium. 

In the case of 5 -nucleotidase as for glucose-6-phosphatase, the enzyme is inseparably bound with phospholipid and the use of detergents such as deoxycholate merely serve to keep the dissociated enzyme and phospholipid units in a state of homogeneous dispersion. Evidence for this is based on the fact that the subsequent removal of detergent by dialysis resulted in the appearance of membranous vesicles as seen under the electron microscope (Song et al., 1967). 

A sigmoid pH-rate curve is obtained characteristic of an enzyme function of pKaz. There is a very rapid production of alcohol, P, and a subsequent slower rate idien enzyme concentration, B, is deficient. This can be explained 5 on the premise that the regeneration of enzyme, E, along with the acyl moiety, Pg from a partially dissociated enzyme c< nplex ES becomes rate determining... 

Michaelis constant An experimentally determined parameter inversely indicative of the affinity of an enzyme for its substrate. For a constant enzyme concentration, the Michaelis constant is that substrate concentration at which the rate of reaction is half its maximum rate. In general, the Michaelis constant is equivalent to the dissociation constant of the enzyme-substrate complex. 

Although extraction of lipids from membranes can be induced in atomic force apparatus (Leckband et al., 1994) and biomembrane force probe (Evans et al., 1991) experiments, spontaneous dissociation of a lipid from a membrane occurs very rarely because it involves an energy barrier of about 20 kcal/mol (Cevc and Marsh, 1987). However, lipids are known to be extracted from membranes by various enzymes. One such enzyme is phospholipase A2 (PLA2), which complexes with membrane surfaces, destabilizes a phospholipid, extracts it from the membrane, and catalyzes the hydrolysis reaction of the srir2-acyl chain of the lipid, producing lysophospholipids and fatty acids (Slotboom et al., 1982 Dennis, 1983 Jain et al., 1995). SMD simulations were employed to investigate the extraction of a lipid molecule from a DLPE monolayer by human synovial PLA2 (see Eig. 6b), and to compare this process to the extraction of a lipid from a lipid monolayer into the aqueous phase (Stepaniants et al., 1997). 

Dinitrogen has a dissociation energy of 941 kj/mol (225 kcal/mol) and an ionisation potential of 15.6 eV. Both values indicate that it is difficult to either cleave or oxidize N2. For reduction, electrons must be added to the lowest unoccupied molecular orbital of N2 at —7 eV. This occurs only in the presence of highly electropositive metals such as lithium. However, lithium also reacts with water. Thus, such highly energetic interactions ate unlikely to occur in the aqueous environment of the natural enzymic system. Even so, highly reducing systems have achieved some success in N2 reduction even in aqueous solvents. 

Enzyme Inhibition. En2yme inhibitors (qv) are reagents that bind to the enzyme and cause a decrease in the reaction rate. Irreversible inhibitors bind to the enzyme by an irreversible reaction, and consequendy cannot dissociate from the enzyme or be removed by dilution or dialysis. Examples of irreversible inhibitors are nerve gases such as diisopropylphosphoduoridate [55-91-4] (DEP). 

Enzyme Kineties The enzyme E and the reactant S are assumed to form a complex ES that then dissociates into product P and uncombined enzyme. 

More than 30 years ago Jacob and Monod introduced the Escherichia coli lac operon as a model for gene regulation. The lac repressor molecule functions as a switch, regulated by inducer molecules, which controls the synthesis of enzymes necessary for E. coli to use lactose as an energy source. In the absence of lactose the repressor binds tightly to the operator DNA preventing the synthesis of these enzymes. Conversely when lactose is present, the repressor dissociates from the operator, allowing transcription of the operon. 

The substrate concentration when the half maximal rate, (Vmax/2), is achieved is called the Km. For many simple reactions it can easily be shown that the Km is equal to the dissociation constant, Kd, of the ES complex. The Km, therefore, describes the affinity of the enzyme for the substrate. For more complex reactions, Km may be regarded as the overall dissociation constant of all enzyme-bound species. 

The Michaelis constant has the units of a dissociation constant however, the dissociation constant of the enzyme—substrate complex is k dk, which is not equal to Km unless k 2-... 

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