Enzyme 440 Subject

Deriving a Rate Equation for Competitive Inhibition The rate equation for an enzyme subject to competitive inhibition is... 

Kletzien, R.F., Harris, P.K., FoeUmi, L.A. (1994) Glucose 6-phosphate dehydrogenase a housekeeping enzyme subject to tissue-specific regulation by hormones, nutrients, and oxidant stress. FASEB J. 8, 174-181. 

The regulatory mechanisms described above modify the activity of existing enzyme molecules. However, cells can also regulate the amount of enzyme present—usually by altering the rate of enzyme synthesis. The increased (induction) or decreased (repression) of enzyme synthesis leads to an alteration in the total population of active sites. [Note The efficiency of existing enzyme molecules is not affected.] Enzymes subject to regulation of synthesis are often those that are needed at only one stage of development or under selected... 

Diffusive and electrostatic effects with insolu-bilized enzymes subject to substrate inhibition (with M.L. Shuler and H.M. Tsuchiya). J. Theor. Biol. 41, 347-356 (1973). 

Enzymes subject to regulation are a select few of the total enzymes in a cell and are so subject for two reasons. First, it is not necessary to regulate the activity of every enzyme to achieve the desired level of control. In many cases, an entire metabolic pathway can be controlled by regulating only the enzyme that catalyzes the first step in the pathway (see chapter 11). Second, elaborate structural properties are required to create an enzyme that can be regulated. 

An even more serious disadvantage of this technique is that it often impairs the biological activity of the modified protein. The activities of hen s egg lysozyme (EC 3.2.1.17) and alpha amylase from Aspergillus oryzae (EC 3.2.1.1) were lessened by diazo coupling of glycosides or aniline.12 Whether the decrease in activity was due to the modification of critical residues, or to the introduction of aromatic structures, is not yet clear however, enzymes subjected to the diazocoupling conditions in the absence of the diazonium salts retained their activity, implying that the reaction conditions themselves were not responsible for the loss of activity. 

Figure 18.2 The glycolysis pathway. The AGq values for each reaction are given in kilojoules. Enzymes subject to various regulatory factors are indicated by jagged boxes. (Reproduced by permission from Wills ED. Biochemical Basis of Medicine Bristol Wright, 1985, p. 584.)...

The glycogenesis and glycogenolysis enzymes subject to hormonal control are glycogen synthase and phosphorylase, respectively. Briefly, glycogen synthase is inhibited by high cellular cAMP and Ca2+ levels, whereas phorphorylase is stimulated. The mechanisms for accomplishing this are quite complex. 

In this experiment we will examine some of the properties of the aspartate transcarbamylase of Escherichia coli, which is typical of many enzymes subject to feedback inhibition and which has been studied extensively. Aspartate transcarbamylase (ATCase) catalyzes the first reaction unique to the biosynthesis of pyrimidine nucleotides. ATCase is subject to specific inhibition by quite low concentrations of one of its end products, cytidine 5 -triphosphate (CTP). This relationship and two other regulatory interactions important to the control of pyrimidine biosynthesis are summarized in Figure 9-1. 

ATCase and many (but not all) enzymes subject to metabolic regulation have a second interesting property their reaction velocity is not a hyperbolic function of substrate (aspartate) concentration, as... 

C3 or C4 was incubated for 60 minutes at 37 C with the respective enzymes, subjected to tricine-PAGE and Coomassie staining and analyzed by scanning the different bands. The values were obtained after background subtraction and normalization to the intensity of the C3p and C4p bands, respectively, except where marked with an asterisk. 

Directed evolution has emerged, within a short time, as a powerful strategy. The list of enzymes subjected to a targeted improvement is continuously increasing, due to the short time required to achieve the desired... 

For an open enzymatic reaction with an enzyme subject to continuous synthesis and breakdown, Nazarenko and Sel kov (1981) gave a two-dimensional model. Furthermore, Sel kov and Nazarenko (1981) proposed a model with an external perturbation and plotted oscillatory solutions. 

Fig. 5. Proposed regulation of fatty acid oxidation in liver. Stimulation inhibition enzymes subject to regulation. Abbreviations ACC, acetyl-CoA carboxylase CPT, carnitine palmitoyltransferase PK, protein kinase.

We have initially described this trend in three different enzymes subjected to a selection for an increase in six different promiscuous activities. The same trend was identified in other laboratory experiments aimed at increasing promiscuous enzymatic and binding activities of various proteins (see Supplementary Table 8 in Aharoni Averaging eighteen cases in which data was provided for the effect of the selected mutations on... 

The reaction of carbamoyl phosphate with aspartate to produce W-carbamo-ylaspartate is the committed step in pyrimidine biosynthesis. The compounds involved in reactions up to this point in the pathway can play other roles in metabolism after this point, A -carbamoylaspartate can be used only to produce pyrimidines—thus the term committed step. This reaction is catalyzed by aspartate transcarbamoylase, which we discussed in detail in Ghapter 7 as a prime example of an allosteric enzyme subject to feedback regulation. The next step, the conversion of A-carbamoylaspartate to dihydroorotate, takes place in a reaction that involves an intramolecular dehydration (loss of water) as well as cyclization. This reaction is catalyzed by dihydroorotase. Dihydroorotate is converted to orotate by dihydroorotate dehydrogenase, with the concomitant conversion of NAD to NADH. A pyrimidine nucleotide is now formed by the reaction of orotate with PRPP to give orotidine-5 -monophosphate (OMP), which is a reaction similar to the one that takes place in purine salvage (Section 23.8). Orotate phosphoribosyltransferase catalyzes this reaction. Finally, orotidine-5 -phosphate decarboxylase catalyzes the conversion of OMP to UMP... 

Figure 13.3 The regulation of the glycolysis pathway. Glucose-6-phosphate is a noncompetitive inhibitor of hexokinase. Phosphofructokinase and pyruvate kinase are also allosteric enzymes subject to control.

Cataboirte repression Inhibition of enzyme synthesis by increased concentrations of certain metabolic products. Enzymes subject to Cr. are formed in reponse to metabolic events (utilization of new nutrients by catabolic enzymes, synthesis of secondary products in certain developmental phases of microorganisms). C. r. is probably present in all organisms, although the molecular mechanisms are diverse. Examples of C.r. are glucose repression of catabolic enzymes in . coU and the enzymes of secondary metabolism in microorganisms, e.g. those for the s thesis of penicillin, actinomycin and riboflavin. 

As with the bacterial reductases, a complex pattern of activation and inhibition by nucleoside triphosphates has been demonstrated for the tumor reductase dATP inhibits reduction of all four substrates (S2). A similar pattern of nucleotide regulatory effects was found with partly purified reductase from rat embryo extracts (S5). Thus, presuming that analogy with the bacterial ribonucleotide reductases is valid, it would appear that the animal reductases are allosteric enzymes subject to a complicated regulation by nucleotides again, the function of such regulation would seem to be that of ensuring a balanced supply of deoxyribonucleo-tides for DNA synthesis. 

That the effect of cyclic AMP is general to catabolite repression was recently demonstrated by DeCrombrugghe et al. [99]. They surveyed a number of enzymes subject to catabohte repression and found that in all cases catabolite repression could be overcome by cyclic AMP. 

Figure 14.9. Rate data for an enzyme subject to substrate inhibition.

Figure 14.14. Modeling of a two-enzyme system, with one enzyme subject to substrate inhibition (A) and the other not inhibited by substrate ( ) using the data in Fig. 14.9 (O). a) Both enzymes are assumed to have the same and make equal contributions to activity observed at 10 mM [S]. (b) Both enzymes are assumed to have the same Km and the uninhibited enzyme contributes 90% of the activity observed at 10 mM [S]. Additional plots (-h -h -h) in (b) predict the behavior of an enzyme subject to substrate inhibition by binding only one molecule to S to form an inactive E S complex with a K value of 1.8 mM (upper curve) or 0.5 mM (lower curve).

The reactions catalyzed by aspartokinase (1) and aspartate semialdehyde dehydrogenase (2) are utilized for the synthesis of all pathway products, including threonine. Regulation of aspartokinase activity is, therefore, considered to be of central importance in the overall control of the pathway. Two classes of differentially regulated isozymes of aspartokinase have been isolated from plants. One class is comprised of enzymes subject to feedback inhibition by threonine the other encompasses those inhibited by lysine, or by lysine and 5 -adenosylmethionine. Examples of each class have been isolated from several... 

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