Intracellular regulation of enzyme activity

As discussed in section 2.3.3, the rate at which an enzyme catalyses a reaction increases with increasing concentration of substrate, until the enzyme is more or less saturated. This means that an enzyme that has a high Km relative to the usual concentration of its substrate will be sensitive to changes in substrate availability. By contrast, an enzyme that has a low Km relative to the usual concentration of its substrate will act at a more or less constant rate regardless of changes in substrate availability.

The availability of substrate may be regulated by uptake from the bloodstream — for example, muscle and adipose tissue only take up glucose to any significant extent in response to insulin. In the absence of insulin the glucose transporters are in intracellular vesicles; in response to insulin these vesicles migrate to the cell surface and fuse with the cell membrane, revealing active glucose transporters. Similarly, fatty acid oxidation is controlled by the availability of fatty acyl CoA in the mitochondrial matrix, and this is regulated by the activity of carnitine palmitoyl transferase (section 5.5.1).

10.2.1 allosteric modification of THE activity of REGuLAToRY ENzYMEs

Allosteric regulation of enzyme activity is due to reversible, non-covalent, binding of effectors to regulatory sites, leading to a change in the conformation of the active site. This may result in either increased catalytic activity (allosteric activation) or decreased catalytic activity (allosteric inhibition). Enzymes that are subject to allosteric regulation are usually multiple subunit proteins.

Many enzymes that are subject to allosteric regulation have two interconvertible conformations:

  • A relaxed (R) form, which binds substrates well, and therefore has high catalytic activity. Allosteric activators bind to, and stabilize, the active R form of the enzyme.
  • A tense (T) form, which binds substrates poorly and therefore has low catalytic activity. Allosteric inhibitors bind to, and stabilize, the less active T form of the enzyme.

Compounds that act as allosteric inhibitors are commonly end-products of the pathway, and this type of inhibition is known as end-product inhibition. The decreased rate of enzyme activity results in a lower rate of formation of a product that is present is adequate amounts.

Compounds that act as allosteric activators of enzymes are often precursors of the pathway, so this is a mechanism for feed-forward activation, increasing the activity of a controlling enzyme in anticipation of increased availability of substrate.

As discussed in section 2.3.3.3, enzymes that consist of multiple subunits frequently display cooperativity between the subunits, so that binding of substrate to the active site of one subunit leads to conformational changes that enhance the binding of substrate to the other active sites of the complex. This again is allosteric activation of the enzyme, in this case by the substrate itself. The activity of such cooperative enzymes is more sharply dependent on the concentration of substrate than is the case for enzymes that do not show cooperativity.

As shown in Figure 10.1, an allosteric activator of an enzyme that shows substrate cooperativity acts by decreasing that cooperativity, so that the enzyme has a greater activity at a low concentration of the substrate than would otherwise be the case. Conversely, an allosteric inhibitor of a cooperative enzyme acts by increasing the cooperativity, so that the enzyme has less activity at a low concentration of substrate than it would in the absence of the inhibitor.

10.2.2 CONTROL OF GLYCOLYSIS - THE ALLOSTERIC REGULATION OF PHOSPHOFRUCTOKINASE

The reaction catalysed by phosphofructokinase in glycolysis, the phosphorylation of fructose 6-phosphate to fructose 1,6-bisphosphate (see Figure 5.10), is essentially

ro a o a) ro activation due to decreased cooperativity activation due to decreased cooperativity

Activateur Reaction Enzymatique

substrate concentration

Figure 10.1 Allosteric inhibition and activation of an enzyme showing subunit cooperativity.

substrate concentration

Figure 10.1 Allosteric inhibition and activation of an enzyme showing subunit cooperativity.

irreversible. In gluconeogenesis, the hydrolysis of fructose 1,6-bisphosphate is catalysed by a separate enzyme, fructose bisphosphatase. Regulation of the activities of these two enzymes determines whether the overall metabolic flux is in the direction of glycolysis or gluconeogenesis.

Inhibition of phosphofructokinase leads to an accumulation of glucose 6-phosphate in the cell, and this results in inhibition of hexokinase, which has an inhibitory binding site for its product. The result of this is a decreased rate of entry of glucose into the glycolytic pathway in tissues other than the liver, which contains glucokinase as well as hexokinase (section 5.3.1), and glucokinase is not inhibited by glucose 6-phosphate. This means that, despite inhibition of glucose utilization as a metabolic fuel, liver can take up glucose for synthesis of glycogen (section 5.6.3).

10.2.2.1 Feedback control of phosphofructokinase

Phosphofructokinase is allosterically inhibited by ATP binding at a regulatory site that is distinct from the substrate binding site for ATP As shown in Figure 10.2, at physiological intracellular concentrations of ATP phosphofructokinase has very low activity and a more markedly sigmoid dependency on the concentration of its substrate. This can be considered to be end-product inhibition, as ATP can be considered to be an end-product of glycolysis.

When there is a requirement for increased glycolysis, and hence increased ATP production, this inhibition is relieved, and there may be a 1000-fold or higher increase in glycolytic flux in response to increased demand for ATP. However, there is less than a 10% change in the intracellular concentration of ATP. Figure 10.3 shows the inhibition of phosphofructokinase; a 10% change would not have a significant effect £

re P

[fructose 6-phosphate], mmol /L

Figure 10.2 ATP.

The substrate dependence of phosphofructokinase at low and physiological concentrations of

Phosphofructokinase

Figure 10.3 The inhibition of phosphofructokinase by ATP, and relief of inhibition by 5-AMP

u ra

Figure 10.3 The inhibition of phosphofructokinase by ATP, and relief of inhibition by 5-AMP

on the activity of the enzyme. What happens is that as the concentration of ADP begins to increase, so adenylate kinase catalyses the reaction:

AMP acts as an intracellular signal that energy reserves are low and ATP formation must be increased. It binds to phosphofructokinase and both reverses the inhibition caused by ATP and increases the cooperativity between the subunits, so that the enzyme has greater affinity for fructose 6-phosphate. AMP also binds to fructose 1,6-bisphosphatase, reducing its activity.

Citrate, which can also be considered to be an end-product of glycolysis, also inhibits phosphofructokinase, by enhancing the inhibition by ATP In muscle, creatine phosphate (section 3.2.3.1) has a similar effect. Phosphoenolpyruvate, which is synthesized in increased amounts for gluconeogenesis (section 5.7), also inhibits phosphofructokinase.

10.2.2.2 Feed-forward control of phosphofructokinase

High intracellular concentrations of fructose 6-phosphate activate a second enzyme, phosphofructokinase-2, which catalyses the synthesis of fructose 2,6-bisphosphate from fructose 6-phosphate (Figure 10.4). Fructose 2,6-bisphosphate is an allosteric activator of phosphofructokinase and an allosteric inhibitor of fructose 1,6-bisphosphatase. It thus acts to both increase glycolysis and inhibit gluconeogenesis. This is feed-forward control — allosteric activation of phosphofructokinase because there is an increased concentration of substrate available.

Glycolysis Fructose Phosphate

fructose 2,6-bisphosphate

Figure 10.4 The role of 2,6-fructose bisphosphate in regulation ofphosphofructokinase.

fructose 2,6-bisphosphate

Figure 10.4 The role of 2,6-fructose bisphosphate in regulation ofphosphofructokinase.

Phosphofructokinase-2 is an interesting enzyme, in that it is a single protein with two catalytic sites. One site is a kinase that catalyses the phosphorylation of fructose 6-phosphate to fructose 2,6-bisphosphate while the other is a phosphatase that catalyses the hydrolysis of fructose 2,6-bisphosphate to fructose 6-phosphate and inorganic phosphate. A single regulatory site controls the activity of the two catalytic sites in opposite directions. In response to glucagon (which stimulates gluconeogenesis and inhibits glycolysis; section 10.3), the kinase activity is decreased and the phosphatase activity increased. This results in a low concentration of fructose 2,6-bisphosphate, and hence decreased activity of phosphofructokinase and increased activity of fructose 1,6-bisphosphatase.

10.2.2.3 Substrate cycling

A priori it would seem sensible that the activities of opposing enzymes such as phosphofructokinase and fructose 1,6-bisphosphatase should be regulated in such a way that one is active and the other inactive at any time. If both were active at the same time then there would be cycling between fructose 6-phosphate and fructose 1,6-bisphosphate, with hydrolysis of ATP — a so-called futile cycle.

What is observed is that both enzymes are indeed active to some extent at the same time, although the activity of one is greater than the other, so there is a net metabolic flux. One function of such substrate cycling is thermogenesis — deliberate hydrolysis of ATP for heat production. It is not known to what extent substrate cycling can be increased to enhance thermogenesis (which is normally mediated by uncoupling of electron transport and oxidative phosphorylation; section 3.3.1.4). However, it is noteworthy that the honey bee, which does not exhibit significant substrate cycling, cannot fly in cold weather, whereas the bumble bee, which has adaptive substrate cycling, can initiate thermogenesis and so fly in cold weather.

Substrate cycling also provides a means of increasing the sensitivity and speed of metabolic regulation. The increased rate of glycolysis in response to a need for ATP for muscle contraction would imply a more or less instantaneous 1000-fold increase in phosphofructokinase activity if phosphofructokinase were inactive and fructose 1,6-bisphosphatase active. If there is moderate activity of phosphofructokinase, but greater activity of fructose 1,6-bisphosphatase, so that the metabolic flux is in the direction of gluconeogenesis, then a more modest increase in phosphofructokinase activity and decrease in fructose 1,6-bisphosphatase activity will achieve the same reversal of the direction of flux.

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Responses

  • helen
    Which figure shows the effect of increased demand?
    7 years ago

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