Enzyme regulatory

Regulatory Enzymes.—Studies of two multi-subunit regulatory enzymes, glutamine synthetase and aspartate transcarbamylase are reported. 

Studies of the regulatory enzyme aspartate transcarbamylaso are further advanced. This enzyme catalyses the first unique reaction with th pyrimidine biosynthetic pathway. The X-ray studies of Wiley et al. show 

One of the most striking facts common to many biosynthetic systems is that enzymes that catalyze the first step in a given synthetic pathway are reversibly inhibited by the end product of the same pathway. This can be presented schematically as follows  

Perhaps the best-studied regulatory enzyme of this kind is aspartate transcarbamoy-lase (ATCase), which catalyzes the first steps in the biosynthetic pathway leading to uridine and cytidine nucleotides. Today, a great amount of information, structural, thermodynamical, and kinetic, is available on this system. Specifically it is known that cytidine triphosphate (CTP) inhibits ATCase. (Other molecules, such as ATP, activate the same enzyme. We shall focus, in this section, on inhibitory effectors only.) 

There are many possibilities by which the end product B can deactivate the first enzyme. For instance, B can bind competitively to the same active site on which the substrate A also binds. Or B can bind to a different, allosteric site and mediate its effect 

Suppose that we use some measure of the activity of the enzyme as a function of the inhibitor concentration p. If B binds competitively to the enzyme Ei, then the activity of the enzyme will fall rapidly with Pb A schematic drawing is given in Fig. 3.20a. 

Clearly, if we are interested in switching the enzyme on and off by manipulating the concentration of B between p = 0 and pi, then this mechanism will answer our requirements and a competitive binding of B to the active site will work. 

Although the kinetic analysis of enzyme-catalyzed reactions has been illustrated by appHcation of the Michaelis—Menten model, not all enzymes react in a way that follows this type of behavior. Enzymes that catalyze 

For an enzyme that follows MichaeHs—Menten kinetics, R = SI. For a regulatory enzyme that gives a sigmoidal rate plot, Rj 81 if the enzyme is exhibiting positive cooperativity, a term that means that the substrate and enzyme bind in such a way that the rate increases to a greater extent with increasing [S] than the MichaeHs—Menten model predicts. Cases with R-s 81 indicate negative cooperativity so that the catalytic effect becomes less than that found in MichaeHs—Menten kinetics. In these cases, kinetic analysis is usually carried out by means of the HiU equation. 

Taking the logarithm of both sides of this equation gives 

Therefore, a graph showing log (R/(Rmax R)) versus log [S] should give a straight line having a slope of n and an intercept of—log K.. Such a graph is 

It should be pointed out that sigmoidal rate plots are sometimes observed for reactions of solids. One of the rate laws used to model such reactions is the Prout-Tompkins equation, the left-hand side of which contains the function ln(a/ (1 — a)) where a is the fraction of the sample reacted (see Section 7.4). The left-hand sides of Eqs. (6.72) and (7.68) have the same form, and both result in sigmoidal rate plots. These cases illustrate once again how gready different types of chemical processes can give rise to similar rate expressions. 

The simplest form of regulation of a metabolic pathway is the inhibition of an enzyme by the product of the pathway. In Fig. 9-5, the E, s denote enzymes, A and B are metabolites, and the circled minus sign indicates inhibition. If there were no inhibitor of the enzyme (E,) acting on A, the concentration of B would depend entirely on its rate of synthesis or utilization. If the rate of utilization of B decreased or B was supplied from an outside source, its concentration would rise, perhaps even to toxic levels. However, if B is an inhibitor of the first enzyme, then as its concentration rises, the extent of inhibition will increase and its rate of synthesis will decrease. This effect is called feedback inhibition or negative feedback control it is a concept also used in describing electronic circuits. 

Control in branched metabolic pathways is more complex. Consider the metabolic scheme in Fig. 9-6. Here, B reacts with C, and D is produced further along the pathway for most effective control of D, B should inhibit the first enzyme (E,) and C should activate it. In this case, if B is supplied from an external source so that B C, then B would inhibit its own synthesis from A and the concentration of B and C would tend to become equal. 

Alternatively, if Cs B, then C would activate the production of B and this again would tend to equalize the concentrations of B and C. This activation by C is usually the result of C competing for the same binding site as B on E[, and thus reducing the inhibition by B. The first enzyme of pyrimidine synthesis, aspartate carbamoyltransferase, in E. coli, is subject to this type of control (Example 8.5 Chap. 15) in this case B is CTP, C is ATP, and D is the nucleic acids. 

Usually effector molecules bear little structural resemblance to the substrates of the enzymes they control. The control is therefore not likely to be due to binding at the active site but at an alternative site, the allosteric site. The effect on the reaction at the active site is mediated by conformational changes in the protein. 

If an effector of an enzyme is also the substrate, it is called a homotropic effector if it is a nonsubstrate, it is called heterotropic. 

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If the kinetics of the reaction disobey the Michaelis-Menten equation, the violation is revealed by a departure from linearity in these straight-line graphs. We shall see in the next chapter that such deviations from linearity are characteristic of the kinetics of regulatory enzymes known as allosteric enzymes. Such regulatory enzymes are very important in the overall control of metabolic pathways. 

Enzymes such as enzyme 1, which are subject to feedback regulation, represent a distinct class of enzymes, the regulatory enzymes. As a class, these enzymes have certain exceptional properties ... 

Inhibition of a regulatory enzyme by a feedback inhibitor does not conform to any normal inhibition pattern, and the feedback inhibitor F bears little structural similarity to A, the substrate for the regulatory enzyme. F apparently acts at a binding site distinct from the substrate-binding site. The term allosteric is apt, because F is sterically dissimilar and, moreover, acts at a site other than the site for S. Its effect is called allosteric Inhibition. 

The search for inhibitors of this pathway began with the first key regulatory enzyme, HMG CoA reductase. Several clinically useful inhibitors of HMG CoA reductase are now known. One of the most successful, Mevacor, produced by Merck, is one of the pharmaceutical industry s best selling products. However, the problem with inhibiting a branched biosynthetic pathway at an early point is that the biosynthesis of other crucial biomolecules may also be inhibited. Indeed, there is some evidence that levels of ubiquinone and the dolichols are affected by some HMG CoA reductase inhibitors. Consequently, efforts have recently been directed towards finding inhibitors of squalene synthase, the enzyme controlling the first step on the route to cholesterol after the FPP branch point. 

The metabolic control is exercised on certain key regulatory enzymes of a pathway called allosteric enzymes. These are enzymes whose catalytic activity is modulated through non-covalent binding of a specific metabolite at a site on the protein other than the catalytic site. Such enzymes may be allosterically inhibited by ATP or allosterically activated by ATP (some by ADP and/or AMP). 

Fructose-2,6-bisphosphatase, a regulatory enzyme of gluconeogenesis (Chapter 19), catalyzes the hydrolytic release of the phosphate on carbon 2 of fructose 2,6-bisphosphate. Figure 7-8 illustrates the roles of seven active site residues. Catalysis involves a catalytic triad of one Glu and two His residues and a covalent phos-phohistidyl intermediate. 

Regulation of the overall flux through a pathway is important to ensure an appropriate supply, when required, of the products of that pathway. Regulation is achieved by control of one or more key reactions in the pathway, catalyzed by regulatory enzymes. The physicochemical factors that control the rate of an... 

Human subjects degrade 1-2% of their body protein daily at rates that vary widely between proteins and with physiologic state. Key regulatory enzymes often have short half-lives. 

ALA Synthase Is the Key Regulatory Enzyme in Hepatic Biosynthesis of Heme... 

IkB kinase-p is a key regulatory enzyme in the NF-kB pathway, and inhibition of this enzyme has the potential for yielding treatments for inflammatory and autoimmune diseases. Morwick et al. [53] report on the optimization of a pM IKKp inhibitor with low aqueous solubility, moderate human liver microsome stability, and inhibition of several CYPs (3A4, 2C9, 1A2) with pM potencies. Modulation of the thiophene core (other thiophene isomer, pyrimidine and oxazole) produces compounds of similar potency to the hit. Fusing the 5-phenyl moiety to the thiophene to form a thieno[2,3-b]pyridine core increases aqueous solubility of the series as well as reduces the CYP liability. While the optimized compound still shows pM IKK(S potency, the aqueous solubility, HLM stability and CYP profiles are much improved. A pharmacophore model was generated that enabled scaffold hopping to yield this new chemotype (Scheme 7). 

Phosphofructokinase (PFK) is a key regulatory enzyme of glycolysis that catalyzes the conversion of fructose-6-phosphate to fructose-1,6-diphosphate. The active PFK enzyme is a homo- or heterotetrameric enzyme with a molecular weight of 340,000. Three types of subunits, muscle type (M), liver type (L), and fibroblast (F) or platelet (P) type, exist in human tissues. Human muscle and liver PFKs consist of homotetramers (M4 and L4), whereas red blood cell PFK consists of five tetramers (M4, M3L, M2L2, ML3, and L4). Each isoform is unique with respect to affinity for the substrate fructose-6-phosphate and ATP and modulation by effectors such as citrate, ATP, cAMP, and fructose-2,6-diphosphate. M-type PFK has greater affinity for fructose-6-phosphate than the other isozymes. AMP and fructose-2,6-diphosphate facilitate fructose-6-phosphate binding mainly of L-type PFK, whereas P-type PFK has intermediate properties. 

Taylor, S. S., Buechler, J. A. and Yonemoto, W. cAmp-depen-dent protein kinase framework for a diverse family o Regulatory enzymes. Annu. Rev. Biochem. 59 971-1005,1990. 

The PDHC catalyzes the irreversible conversion of pyruvate to acetyl-CoA (Fig. 42-3) and is dependent on thiamine and lipoic acid as cofactors (see Ch. 35). The complex has five enzymes three subserving a catalytic function and two subserving a regulatory role. The catalytic components include PDH, El dihydrolipoyl trans-acetylase, E2 and dihydrolipoyl dehydrogenase, E3. The two regulatory enzymes include PDH-specific kinase and phospho-PDH-specific phosphatase. The multienzyme complex contains nine protein subunits, including... 

NAGY, P.L., MAROLEWSKI, A., BENKOVIC, S.J, ZALKIN, H., Formyltetrahydro-folate hydrolase, a regulatory enzyme that functions to balance pools of tetrahydrofolate and one-carbon tetrahydrofolate adducts in Escherichia coli, J. Bacteriol., 1995, 177, 1292-1298. 

Phosphoryl group transfer reactions add or remove phosphoryl groups to or from cellular metabolites and macromolecules, and play a major role in biochemistry. Phosphoryl transfer is the most common enzymatic function coded by the yeast genome and, in addition to its importance in intermediary metabolism (see Chapter 5), the reaction is catalysed by a large number of central regulatory enzymes that are often part of signalling cascades, such as protein kinases, protein phosphatases, ATPases and GTPases. 

Glucagon stimulation of liver cells in particular leads to phosphorylation of regulatory enzymes whereas insulin has the opposite effect. So, after a meal, we would expect glycolysis and glycogen synthesis to operate very efficiently so the control enzymes will be dephosphorylated. 

Recently it has been shown that when a protein molecule contains two or more sub-units, which may or may not be identical, the quaternary structure or relative positions of the sub-units is important for function. Many regulatory enzymes are examples of this type of protein 26, 27). 

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