Citrate enzyme regulation

Citrate synthase, isocitrate dehydrogenase and oxogluta-rate dehydrogenase are key enzymes regulating the flux through the cycle all three catalyse non-equilibrium reactions (Chapter 3). 

Thus far we have discussed the two most important enzymes regulating the supplies of acetyl-CoA and oxaloacetate for the TCA cycle. This leaves us with the three enzymes, all within the cycle, that regulate the activity of the cycle. The first of these, citrate synthase, catalyzes the formation of citrate from acetyl-CoA and oxaloacetate. Regulation, by energy charge and other parameters, of the rate of glycolysis and of the pyruvate dehydrogenase reaction play... 

Binding of ADP or AMP aUostericaUy activates phosphofructokinase, but high concentrations of ATP are inhibitory. At first sight this seems to be a contradictory response elicited by ATP since it is a substrate for the enzyme. However, careful analysis of the relationship between the activity of phosphofructokinase and ATP concentration shows that there is always sufficient ATP in the normal cell for the reaction to proceed, but that ATP inhibition becomes significant only when its concentration is very high. Fructose 2,6-bisphosphate is an inhibitor, as is citrate. The regulation of the concentration of fructose 2,6-bisphosphate is via a phosphoiylation-dephosphorylation process as shown in Fig. 11-12. 

Citrate synthase is the first step in this metabolic pathway, and as stated the reaction has a large negative AG°. As might be expected, it is a highly regulated enzyme. NADH, a product of the TCA cycle, is an allosteric inhibitor of citrate synthase, as is succinyl-CoA, the product of the fifth step in the cycle (and an acetyl-CoA analog). 

The hydrolysis of fructose-1,6-bisphosphate to fructose-6-phosphate (Eigure 23.7), like all phosphate ester hydrolyses, is a thermodynamically favorable (exergonic) reaction under standard-state conditions (AG° = —16.7 kj/mol). Under physiological conditions in the liver, the reaction is also exergonic (AG = —8.6 kJ/mol). Fructose-1,6-bisphosphatase is an allosterically regulated enzyme. Citrate stimulates bisphosphatase activity, hut fructose-2,6-bisphosphate is a potent allosteric inhibitor. / MP also inhibits the bisphosphatase the inhibition by / MP is enhanced by fructose-2,6-bisphosphate. 

Because this enzyme catalyzes the committed step in fatty acid biosynthesis, it is carefully regulated. Palmitoyl-CoA, the final product of fatty acid biosynthesis, shifts the equilibrium toward the inactive protomers, whereas citrate, an important allosteric activator of this enzyme, shifts the equilibrium toward the active polymeric form of the enzyme. Acetyl-CoA carboxylase shows the kinetic behavior of a Monod-Wyman-Changeux V-system allosteric enzyme (Chapter 15). 

Lipogenesis is regulated at the acetyl-CoA carboxylase step by allosteric modifiers, phosphorylation/de-phosphorylation, and induction and repression of enzyme synthesis. Citrate activates the enzyme, and long-chain acyl-CoA inhibits its activity. Insulin activates acetyl-CoA carboxylase whereas glucagon and epinephrine have opposite actions. 

At least two enzymes compete for acetyl-CoA - the citrate synthase and 3-ke-tothiolase. The affinities of these enzymes differ for acetyl-CoA (Table l),and at low concentrations of it the citrate synthase reaction tends to dominate, provided that the concentration of 2/H/ is not inhibiting. The fine regulation of the citrate synthases of various poly(3HB) accumulating bacteria has been studied [ 14, 47, 48]. They appear to be controlled by cellular energy status indicators (ATP, NADH, NADPH) and/or intermediates of the TCA cycle. The 3-ketothio-lase has also been investigated [10-14,49, 50]. This enzyme is, above all, inhibited by CoASH [10,14,49]. This important feature will be further considered below. 

The theory of regulation of the cycle is as follows. First, an increase in oxaloacetate concentration increases the activity of citrate synthase and hence the cycle. The concentration of oxaloacetate is regulated by the activity of the enzyme pyruvate carboxylase, which catalyses the reaction ... 

Why did nature use an Fe-S cluster to catalyze this reaction, when an enzyme such as fumarase can catalyze the same type of chemistry in the absence of any metals or other cofactors One speculation would be that since aconitase must catalyze both hydrations and dehydrations, and bind substrate in two orientations, Fe in the comer of a cubane cluster may provide the proper coordination geometry and electronics to do all of these reactions. Another possibility is that the cluster interconversion is utilized in vivo to regulate enzyme activity, and thus, help control cellular levels of citrate. A third, but less likely, explanation is that during evolution an ancestral Fe-S protein, whose primary function was electron transfer, gained the ability to catalyze the aconitase reaction through random mutation. 

The most important factor in the regulation of the cycle is the NADH/NAD ratio. In addition to pyruvate dehydrogenase (PDH) and oxoglu-tarate dehydrogenase (ODH see p. 134), citrate synthase and isodtrate dehydrogenase are also inhibited by NAD deficiency or an excess of NADH+HT With the exception of isocitrate dehydrogenase, these enzymes are also subject to product inhibition by acetyl-CoA, suc-cinyl-CoA, or citrate. 

Acetyl-CoA carboxylase is also regulated by covalent modification. Phosphorylation, triggered by the hormones glucagon and epinephrine, inactivates the enzyme and reduces its sensitivity to activation by citrate, thereby slowing fatty acid synthesis. In its active (dephosphorylated) form, acetyl-CoA carboxylase polymerizes into long filaments (Fig. 21-1 lb) phosphorylation is accompanied by dissociation into monomeric subunits and loss of activity. 

Heterotropic effectors The effector may be different from the substrate, in which case the effect is said to be heterotropic. For example, consider the feedback inhibition shown in Figure 5.17. The enzyme that converts A to B has an allosteric site that binds the end-product, E. If the concentration of E increases (for example, because it is not used as rapidly as it is synthesized), the initial enzyme in the pathway is inhibited. Feedback inhibition provides the cell with a product it needs by regulating the flow of substrate molecules through the pathway that synthesizes that product. [Note Heterotropic effectors are commonly encountered, for example, the glycolytic enzyme phosphofructokinases allosterically inhibited by citrate, which is not a substrate for the enzyme (see p. 97).]... 

The reaction catalyzed by phosphofructokinase A. is activated by high concentrations of ATP and citrate. B. uses fructose 1-phosphate as substrate. C. is the regulated reaction of the glycolytic pathway. D. is near equilibrium in most tissues. E. is inhibited by fructose 2,6-bisphosphate. Correct answer = C. Phosphofructokinase is the pace-setting enzyme of glycolysis. It is inhibited by ATP and citrate, uses fructose 6-phosphate as substrate, and catalyzes a reaction that is far from equilibrium. The reaction is activated by fructose 2,6-bisphosphate. 

The regulated step in fatty acid synthesis (acetyl CoA - malonyl CoA) is catalyzed by acetyl CoA carboxylase, which requires biotin. Citrate is the allosteric activator, and long-chain fatty acyl CoA is the inhibitor. The enzyme can also be activated in the presence of insulin and inactivated in the presence of epinephrine or glucagon. 

ADP, acetyl-CoA, succinyl-CoA, and citrate. The major known sites for regulation of the cycle include two enzymes outside the cycle (pyruvate dehydrogenase and pyruvate carboxylase) and three enzymes inside the cycle (citrate synthase, isocitrate dehydrogenase, and a-ketoglutarate dehydrogenase). All of these sites of regulation represent important metabolic branchpoints. 

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