Enzyme Activity: Allosteric Regulation

Abstract

Cells can respond to changes in their environment by altering the flow through special, regulated metabolic steps performed by allosteric enzymes. These enzymes adopt either an active or inactive conformation in response to binding positive or negative effectors. As the energy difference between the two conformations is normally modest, the binding of a small metabolite ligand is adequate to stabilise the conformation that binds it. Most allosteric enzymes are K‐type, denoting that the principal feature that is altered is their affinity for their substrate, measured as the KM constant. This enables them to become more or less active when the substrate concentration in the cell remains largely constant. A small, but important, subset of enzymes are V‐type, denoting that the important change is in their maximum activity, defined as Vmax. These normally inactive enzymes can increase their activity by up to a million‐fold.

Key Concepts:

  • All protein enzymes are sufficiently flexible to modestly alter their folded structure and experience two or more different conformational states under normal physiological conditions.

  • Such conformational changes may modestly alter the position of amino acids around the catalytic site, thereby making the ability of the substrate to bind at this site more, or less, favourable.

  • The change in the free energy for such different conformations is quite modest (normally in the range of 2–7 kcal mol−1). Then, if one of these conformations has an appropriately formed binding site for a regulatory ligand, the binding energy with such a ligand binding with simple noncovalent interactions (normally in the range of 2–7 kcal mol−1) is adequate to stabilise that particular conformation, and make it more abundant in the cell.

  • For about a third of all enzymes there exist one or more cellular metabolite(s) that can bind to and stabilise either the more active or the less active conformation.

  • Many allosteric enzymes may also be regulated by being covalently modified, via the attachment of some chemical group. Though more than 20 different types of such regulatory adducts have been observed, phosphorylation is most common, being used with almost half of all allosteric enzymes.

  • Allosteric enzymes are defined as K‐type (approximately 30% of all enzymes) or V‐type (less than 1% of enzymes), depending on whether the major regulatory feature is their change in affinity (KM) or their change in maximum activity (Vmax).

  • More than 90% of K‐type enzymes display positive cooperativity, as they are converted from a fairly inactive T conformation to the active R conformation.

  • A fairly small subset of K‐type enzymes displays negative cooperativity, resulting when many of the catalytic sites, either in a single enzyme oligomer, or in the overall enzyme ensemble, have a normal affinity for the substrate, while the remaining sites have a much lower affinity. This feature greatly extends the concentration range over which the substrate can be used.

Keywords: allosteric; conformation; enzyme; negative cooperativity; positive cooperativity; regulation

Figure 1.

There are three types of enzymes: normal enzymes that have no regulation, except by product inhibition, plus two types of allosteric enzymes that are controlled by activators and inhibitors. Haemoglobin was the original protein defined for allosteric regulation in the binding of oxygen, and is included for comparison. Note that although various icons are used to suggest large conformational changes, there are many allosteric enzymes where no significant change in tertiary structure has been observed.

Figure 2.

Sigmoidal kinetics define an allosteric enzyme with positive cooperativity. The dashed lines indicate the concentration of substrate at which the enzyme has 50% of the maximum activity (kcat), and the intersection on the abscissa indicates the enzyme's binding affinity for the substrate. (a) Normal enzyme (no cooperativity); (b) allosteric enzyme (cooperative kinetics). The upper horizontal line denotes kcat or Vmax.

Figure 3.

Energetics and equilibria for the R and T enzyme conformations. As this is a general depiction, actual energy levels are arbitrary. Abbreviations: T, inactive conformation; R, active conformation; A, activator; I, inhibitor and S, substrate; (a) and (b) depict a system where the T conformation is more stable, whereas in (c) and (d) the R conformation is more stable.

Figure 4.

Examples of (a) no cooperativity, (b) positive cooperativity and (c) negative cooperativity. In (c) if one had many very accurate experimental points, each at a slightly higher concentration, the solid curve obtained would clearly show an inflection as the high affinity sites (KM1) became saturated, and a second hyperbola as the weaker sites (KM2) became saturated, thereby indicating negative cooperativity. However, if one had a small number of experimental data points, with some modest scatter (black dots), one might be tempted to fit a hyperbola to the entire dataset, and interpret this as no cooperativity. Dashed lines indicate 50% binding and this defines KM or K0.5. To define KM1 and KM2 it is assumed that this enzyme has exactly one‐half the sites with high or with low affinity.

Figure 5.

Enzyme kinetics showing negative cooperativity for CTP synthetase. Enzyme velocity was determined as a function of the substrate glutamine, plus increasing concentrations of the activator GTP. Note that the lowest curve is comparable to two hyperbolic curves added together, reflecting the separate KM's for glutamine of 0.08 mM and 0.8 mM. Adapted from Levitzki and Koshland ().

Figure 6.

A simple ligand binding curve: the binding of H+ by bicarbonate anion. (a) Semilog plot and (b) linear plot for the same binding experiment.

Figure 7.

The correlation between change in affinity and change in maximum activity for V‐type enzymes. Red symbols are for human proteases in the blood‐clotting cascade. The blue symbol is for the human G‐protein p21ras. The vertical dashed line represents no change in affinity; the horizontal dashed line represents no change in kcat. Note that Vmax and kcat have the same meaning. For K‐type enzymes kcat is normally fairly constant, and KM changes by less than 10‐fold.

Figure 8.

Models for cooperativity in the binding of oxygen by haemoglobin, a tetramer with four subunits. (a) Concerted or symmetry model where all four subunits must have the same conformation and (b) sequential or induced fit model where subunits independently change conformation as oxygen binds.

Figure 9.

Graphic plots for analysing enzyme kinetics. For each of the four types of graphic analysis: (a) is for noncooperativity; (b), positive cooperativity and (c), negative cooperativity. For the Hill plots, kcat must first be determined with one of the other three plots. The red dashed line has a slope of 1.0. The blue dashed lines indicate the affinity for substrate.

Figure 10.

Structure of phosphofructokinase from B. stearothermophilus. The enzyme is a tetramer and each subunit has two domains. The two substrates, F6P and ATP, define the catalytic site. Two alternate effectors bind at the same regulatory site. Note that the binding of F6P, and also of the effectors, is between subunits.

Figure 11.

Catalytic site of phosphofructokinase. The solid line represents the backbone of the polypeptide chain; dashed lines in red represent noncovalent bonds. The bold dashed line is the interface between subunit 1 and subunit 2 of Figure : (a) T conformation in the absence of ligands and (b) R conformation when F6P and ADP are at the catalytic site. Amino acids 155–162 form the 6F loop. Adapted from Schirmer and Evans ().

Figure 12.

Allosteric regulation as a function of regulatory effectors and available substrate concentrations. Arrows on the abscissa indicate (a) the concentration of substrate equal to the affinity (apparent KM) for different conformations of an allosteric enzyme, and (b) the extent of change in activity that is possible at a fixed, cellular substrate concentration.

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References

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Levitzki A and Koshland DE Jr (1969) Negative cooperativity in regulatory enzymes. Proceedings of the National Academy of Sciences of the USA 62: 1121–1128.

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Schirmer T and Evans PR (1990) Structural basis of the allosteric behaviour of phosphofructokinase. Nature 343: 140–145.

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Further Reading

Perutz M (1990) Mechanisms of Cooperativity and Allosteric Regulation in Proteins. New York: Cambridge University Press.

Traut T (2008) Allosteric Regulatory Enzymes. New York: Springer.

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Traut, Thomas(May 2014) Enzyme Activity: Allosteric Regulation. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0000865.pub3]