Conjugate conjugated enzyme, effect

Many enzymes require additional substances in order to function effectively. Conjugated enzymes require a prosthetic group before they are catalytically active, such groups being covalently or ionically linked to the protein molecule and remaining unaltered at the end of the reaction. Catalase (EC 1.11.1.6), for instance, contains a haem group while ascorbate oxidase (EC 1.10.3.3) contains a copper atom. 

Phenol is a hydrolyzed metabolite of benzene and is itself further hydrolyzed or conjugated to produce other compounds. Therefore, the toxic effects of phenol exposure may be due to a combination of the parent compound and its metabolites. The major tissues in which metabolism appears to occur are the liver, gut, lung, and kidney (Cassidy and Houston 1984 Powell et al 1974 Quebbemann and Anders 1973 Tremaine et al. 1984). Since phenol, benzene, and their major metabolites all seem to compete for the same P450 and conjugating enzymes, metabolic reactions are presumed to be saturable. 

Codeine, one of the principal alkaloids of opium, has an analgesic efficacy much lower than other opioids, due to an extremely low affinity for opioid receptors. It is approximately one-sixth as potent as morphine. It has a low abuse potential. In contrast to other opioids, with the exception of oxycodone, codeine is relatively more effective when administered orally than parenterally. This is due to methylation at the C3 site on the phenyl ring (Figure 7.3), which may protect it from conjugating enzymes. It is used in the management of mild-to-moderate pain, often in combination with non-opioid analgesics, such as aspirin or paracetamol. It is valuable as an antitussive and for the treatment of diarrhoea. Side effects are uncommon and respiratory depression, even with large doses, is seldom a problem. 

Our present approach in the preparation of new dextran-enzyme conjugates is initially to test the standard conditions. If such conditions do not give satisfactory results, either in terms of retention of activity or extent of conjugation, the effect of the three important parameters, pH, temperature and duration of reaction are then investigated to establish appropriate conditions for coupling. In some cases it has also been found useful to examine the effect of these variables on the stability characteristics and other properties of the resulting dextran-enzyme conjugates. 

Effect of Protein Denaturants. Most of the conjugated enzymes we have prepared show greater resistance to inactivation than do the corresponding native enzymes when treated with protein... 

Effect of Carbohydrate on Enzyme-Substrate Interaction. Since many of the enzymes we have conjugated with dextran have macromolecules as their natural substrates, we recognized that the conjugation process might result in unfavorable steric interactions that would impair the ability of the enzymes to interact with such substrates, in the same way that attached carbohydrate affects the susceptibility of the conjugated enzymes to proteolytic attack. We have therefore investigated the effect of carbohydrate on the interaction of conjugated enzymes with substrate. 

Miscellaneous. In addition to the effect of conjugation of a-amylase on its heat stability, stability in denaturants, and stability on cofactor removal, the attachment of dextran also results in improved stability of this acid-labile enzyme at pH values below about 5.0. Studies on the dependence of stability of Bacillus amyloliquefaaiens a-amylase on pH showed that the conjugated enzyme retained 20, 15 and 7% more activity at pH 3.5, 4.0 and 4.5 than did the native enzyme. 

Two "non-effects" of conjugation may also be mentioned briefly. In no case does carbohydrate attachment markedly affect the pH-activity relationship of any of the enzymes we have conjugated. This observation is not surprising since we have always conjugated enzymes with uncharged polysaccharide. It remains to be determined whether attachment of an enzyme to a charged polysaccharide affects its pH optimum. 

A number of enzyme systems have evolved in animals and plants which effectively convert lipophilic xenobiotics to more polar compounds that are efficiently excreted. Phase I enzymes, responsible for oxidation, reduction, and/or hydrolysis, are integrated with phase II or conjugation enzymes for reactions of both types and are normally required for the formation of products polar enough to be readily excreted. The intracellular level of these enzymes, and thus, the capacity for biotransformation, increases in a coordinate fashion in response to exposure to xenobiotic compounds. This response is... 

Benziodarone, a coronary vasodilator, causes in vitro inhibition of the BSP conjugating system. This drug increases the retention of BSP in plasma but has no effect on the retention of phenol dibromophthalein disulfonate, which is not conjugated. Benziodarone probably acts specifically on the conjugating enzyme since it has been shown to inhibit other conjugations with glutathione (B38). 

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