Enzymes, inhibition, substrate properties

A reciprocal plot of the effect of varying concentrations of a noncompetitive inhibitor on enzyme-catalyzed substrate turnover readily reveals the nature and characteristics of this type of inhibition (Fig. 3.6). Notice that in this case, the properties that characterize noncompetitive inhibition are virtually opposite of those that characterize competitive inhibition. With a noncompetitive inhibitor Emax does change but KM is constant. 

Enzyme immobilization has been reported to improve the thermal stability of enzymes (1,2) and may also affect binding of substrates and inhibitors to the enzyme, thereby affecting the Michaelis constant and enzyme inhibition. Several previous studies have considered the advantages of immobilized enzymes with soluble substrates, and a few studies have also investigated the properties of immobilized enzymes with insoluble substrates. The main objective of the present work was to establish the effect of immobilization on the thermal stability of these enzymes, so that they may be used at elevated temperatures without significant activity loss. The immobilization conditions were varied, and their effect on the performance of the immobilized enzymes was analyzed with reference to their physiochemical and structural properties. 

Aspartate transcarbamylase (ATCase) catalyzes the formation of carbamoyl aspartate with CP and aspartic acid as substrates. It is the first specific enzyme for the pyrimidine pathway, and it holds a special place in the historical development of end-product control at this level. The concept of feedback inhibition as an important regulatory mechanism evolved from the initial discovery by Yates and Pardee [90] that CTP is a potent inhibitor of ATCase. It has since developed into a prototype for a regulatory protein with classic allosteric properties. A thorough characterization of the enzyme and its properties has been made through the combined efforts of Gerhart, Pardee, Schachman, and Changeux [91-97]. A summary of these studies follows. 

The kinetic behaviour of a bound enzyme inhibited by the substrate shows multiple steady states within a particular range of concentrations of the substrate in the macroenvironment, provided diffusion to the enzyme is slow. When the substrate has to cross a membrane to reach the enzyme, regulatory schemes showed that the enzyme s activity can be changed dramatically by small variations in substrate concentration, membrane permeability, and the kinetic constants of the enzyme. It was concluded that the regulatory properties of an enzyme in a cell are more effective than suggested by experiments with the enzyme in solution. 

In plants, the (w-hydroxylase system is responsible for synthesis of the o-hydroxy fatty acid components of cutin and suberin. Kolattukudy has studied the reactions in preparations from Vida faba. NADPH and O2 were cofactors and the enzyme showed typical properties of a mixed-function oxidase. However, the involvement of P450 in the plant system is unproven since, although the hydroxylation is inhibited by CO, the inhibition is not reversed by 420-460 nm light (a property typical for cytochrome P450 systems). Where dihydroxy fatty acids are being synthesized for cutin or suberin, the co-hydroxy fatty acid is the substrate for the second hydroxylation. Like tw-oxidation, mid-chain hydroxylation also requires NADPH and O2 and is located in the endoplasmic reticulum. 

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