Modification of enzymes

The commercial exploitation of our increased understanding of protein stmcture will not, of course, be restricted to the pharmaceutical industry. The industrial use of enzymes in the chemical industry, the development of new and more specific pesticides and herbicides, the modification of enzymes in order to change the composition of plant oils and plant carbohydrates are all examples of other commercial developments that depend, in part, on understanding the structure of particular proteins at high resolution. 

It is not only the activity that can be altered by incorporation of noncoded amino acids. Introduction of structures possessing certain chemical functions leads to the possibility of highly regioselective modification of enzymes. For example, selective enzymatic modification of cystein residues with compounds containing azide groups has led to the preparation of enzymes that could be selectively immobilized using click chemistry methods [99]. 

Covalent modification of enzymes (molecular weight of several hundreds or thousands) by the incorporation of inorganic phosphate in the form of P03 (formula weight = 85), seems to represent a small chemical change in the enzyme yet is an important control mechanism of enzyme activity. Explain how phosphorylation can exert its controlling effect on the activity of the enzyme. 

Metabolism is tightly regulated by a number of mechanisms feedback inhibition, compartmentalization, covalent modification of enzymes (e.g., phosphorylation), and hormone action, among others. 

Colman has also produced a definitive account of site-specific modification of enzymes, and her chapter is particularly instructive about the range and utility of reaction types that can be gainfully exploited in affinity labeling experiments. 

Resell, C., Femandez-Lafuente, R. and Gnisan, J.M. (1995) Modification of enzyme properties by the use of inhibitors dnring their stabilisation by multipoint covalent attachment. Biocatalysis and Biotransformation, 12, 67-76. 

In Chapters 13 through 22 we have discussed metabolism at the level of the individual cell, emphasizing central pathways common to almost all cells, prokaryotic and eukaryotic. We have seen how metabolic processes within cells are regulated at the level of individual enzyme reactions, by substrate availability, by allosteric mechanisms, and by phosphorylation or other covalent modifications of enzymes. 

The flow of intermediates through metabolic pathways is controlled by 1bir mechanisms 1) the availability of substrates 2) allosteric activation and inhibition of enzymes 3) covalent modification of enzymes and 4) induction-repression of enzyme synthesis. This scheme may at first seem unnecessarily redundant however, each mechanism operates on a different timescale (Figure 24.1), and allows the body to adapt to a wde variety of physiologic situations. In the fed state, these regulatory mechanisms ensure that available nutrients are captured as glycogen, triacylglycerol, and protein. 

J. Eyzaguirre, ed., Chemical modification of enzymes, active site studies, Ellis Norwood series in biochemistry, Ellis Horwood Ltd. (1987). 

Covalent modifications of enzymes allow a cell to regulate its metabolic activities more rapidly and in much more intricate ways than is possible by changing the absolute concentrations of the same enzymes. They still do not provide truly instantaneous responses to changes in conditions, however, because each modification requires the action of... 

Other methods of stabilization include chemical or carbohydrate modification of enzymes. Modifications of reactive groups on proteins without insolubilization has been used to enhance stability in solution. Grafting of polysaccharides or synthetic polymers, alkalation, acetylation and amino acid modification have all been reported (5)... 

Russel, A. J., and Fersht, A. R. (1987). Rational modification of enzyme catalysis by engineering surface charge. Nature, 328, 496-500. 

What is fast What is slow What is relevant The relaxation time concept of Harder and Roels [148] (Fig. 28) maps typical time constants of microbial and cellular control on the level of modification of enzymes (activation, inhibition, dis/association of subunits, covalent modification or digestion) to the range of ms to s, on the level of regulation of gene expression (induction, repression or derepression of transcription) to min, on the level of population selection and evolution to days and larger units. The examples discussed below will clear up how bioengineering is facing the individual time constants. 

There are two general types of covalent modification of enzymes that regulate their activity. These are the irreversible activation of inactive enzyme precursors, the zymogens, and the reversible interconversion of active and inactive forms of an enzyme. 

Figure 5.15 Role of cAMP in covalent modification of enzyme activity.

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