Garrett R.H., Grisham C.M. - Biochemistry (1999)(2nd ed.)(en)

Effects of I:

Thus, I inhibits association of S and A with R by lowering R0 level. I increases cooperativity of substrate saturation curve. I raises the apparent value of L .

Daniel Koshland has championed the idea that proteins are inherently flexible molecules whose conformations are altered when ligands bind. This notion serves as the fundamental tenet of the “induced-fit hypothesis” discussed earlier. Because this is so, ligand binding can potentially cause conformational changes in the protein. Depending on the nature of these conformational changes, virtually any sort of allosteric interaction is possible. That is, the binding of one ligand could result in conformational transitions in the protein that make it easier or harder for other ligands (of the same or different kinds) to bind. In 1966, Koshland and his colleagues proposed an allosteric model in which ligandinduced conformational changes caused transition to a conformational state with altered affinities. Because ligand binding and conformational transitions were distinct steps in a sequential pathway, the Koshland, Nemethy, Filmer (or KNF) model is dubbed the sequential model for allosteric transitions. The figure depicts the essential features of this model in a hypothetical dimeric protein. Binding of the ligand S induces a conformational change in the subunit to which it binds. Note that there is no requirement for conservation of symmetry here; the two subunits can assume different conformations (represented as a square and a circle). If the subunit interactions are tightly coupled, then binding of S to one subunit could cause the other subunit(s) to assume a conformation having a greater, or a lesser, affinity for S (or some other ligand). The underlying mechanism rests on the fact that the ligand-induced conformational change in one subunit can transmit its effects to neighboring subunits by changing the interactions and alignments of amino acid residues at the interface between subunits. Depending on the relative ligand affinity of the conformation adopted by the neighboring subunit, the overall effect for further ligand binding may be positive, negative, or neutral (figure).

(a) Binding of S induces a conformational change.

(b)

Transmitted

S S conformational

change

If the relative affinities of the various conformations for S are:

positive homotropic effects ensue.

If the relative affinities of the various conformations for S are:

negative homotropic effects are seen.

The Koshland–Nemethy–Filmer sequential model for allosteric behavior. (a) S-binding can, by induced fit, cause a conformational change in the subunit to which it binds. (b) If subunit interactions are tightly coupled, binding of S to one subunit may cause the other subunit to assume a conformation having a greater (positive homotropic) or lesser (negative homotropic) affinity for S. That is, the ligand-induced conformational change in one subunit can affect the adjoining subunit. Such effects could be transmitted between neighboring peptide domains by changing alignments of nonbonded amino acid residues.

K Systems and V Systems

The allosteric model just presented is called a K system because the concentration of substrate giving half-maximal velocity, defined as K0.5, changes in response to effectors (Figure 15.11). Note that Vmax is constant in this system.

An allosteric situation where K0.5 is constant but the apparent Vmax changes in response to effectors is termed a V system. In a V system, all v versus S plots are hyperbolic rather than sigmoid (Figure 15.12). The positive heterotropic effector A activates by raising Vmax, whereas I, the negative heterotropic effector, decreases it. Note that neither A nor I affects K0.5. This situation arises if R and T have the same affinity for the substrate, S, but differ in their catalytic ability and their affinities for A and I. A and I thus can shift the relative T/R distribution. Acetyl-coenzyme A carboxylase, the enzyme catalyzing the committed step in the fatty acid biosynthetic pathway, behaves as a V system in response to its allosteric activator, citrate (see Chapter 25).

K Systems and V Systems Fill Different Biological Roles

The K and V systems have design features that mean they work best under different physiological situations. “K system” enzymes are adapted to conditions in which the prevailing substrate concentration is rate-limiting, as when [S] in vivo K0.5. On the other hand, when the physiological conditions are such that [S] is usually saturating for the regulatory enzyme of interest, the enzyme conforms to the “V system” mode in order to have an effective regulatory response.

15.5 ● Glycogen Phosphorylase: Allosteric Regulation

and Covalent Modification

The Glycogen Phosphorylase Reaction

The cleavage of glucose units from the nonreducing ends of glycogen molecules is catalyzed by glycogen phosphorylase, an allosteric enzyme. The enzymatic reaction involves phosphorolysis of the bond between C-1 of the departing glucose unit and the glycosidic oxygen, to yield glucose-1-phosphate and a glycogen molecule that is shortened by one residue (Figure 15.13). (Because

+A

0

v

+I

[S]

FIGURE 15.12 ● v versus [S] curves for an allosteric “V” system. The V system fits the model of Monod, Wyman, and Changeux, given the following conditions: (1) R and T have the same affinity for the substrate, S. (2) The effectors A and I have different affinities for R and T and thus can shift the relative T/R distribution. (That is, A and I change the apparent value of L.) Assume as before that A binds “only” to the R state and I binds “only” to the T state. (3) R and T differ in their catalytic ability. Assume that R is the enzymatically active form, whereas T is inactive. Because A perturbs the T/R equilibrium in favor of more R, A increases the apparent Vmax. I favors transition to the inactive T state.

Each subunit contributes a tower helix (residues 262 to 278) to the sub- unit–subunit contact interface in glycogen phosphorylase. In the phosphorylase dimer, the tower helices extend from their respective subunits and pack against each other in an antiparallel manner.

Regulation of Glycogen Phosphorylase by Allosteric Effectors

Muscle Glycogen Phosphorylase Shows Cooperativity in Substrate Binding

The binding of the substrate inorganic phosphate (Pi) to muscle glycogen phosphorylase is highly cooperative (Figure 15.16a), which allows the enzyme activity to increase markedly over a rather narrow range of substrate concentration. Pi is a positive homotropic effector with regard to its interaction with glycogen phosphorylase.

ATP and Glucose-6-P A re Allosteric Inhibitors of Glycogen Phosphorylase

ATP can be viewed as the “end product” of glycogen phosphorylase action, in that the glucose-1-P liberated by glycogen phosphorylase is degraded in muscle via metabolic pathways whose purpose is energy (ATP) production. Glucose- 1-P is readily converted into glucose-6-P to feed such pathways. (In the liver, glucose-1-P from glycogen is converted to glucose and released into the bloodstream to raise blood glucose levels.) Thus, feedback inhibition of glycogen phosphorylase by ATP and glucose-6-P provides a very effective way to regulate glycogen breakdown. Both ATP and glucose-6-P act by decreasing the affinity of glycogen phosphorylase for its substrate Pi (Figure 15.16b). Because the binding of ATP or glucose-6-P has a negative effect on substrate binding, these substances act as negative heterotropic effectors. Note in Figure 15.16b that the substrate saturation curve is displaced to the right in the presence of ATP or glu- cose-6-P, and a higher substrate concentration is needed to achieve half-maxi- mal velocity (Vmax/2). When concentrations of ATP or glucose-6-P accumulate to high levels, glycogen phosphorylase is inhibited; when [ATP] and [glucose- 6-P] are low, the activity of glycogen phosphorylase is regulated by availability of its substrate, Pi.

A M P Is an Allosteric Activator of Glycogen Phosphorylase

AMP also provides a regulatory signal to glycogen phosphorylase. It binds to the same site as ATP, but it stimulates glycogen phosphorylase rather than inhibiting it (Figure 15.16c). AMP acts as a positive heterotropic effector, meaning that it enhances the binding of substrate to glycogen phosphorylase. Significant levels of AMP indicate that the energy status of the cell is low and that more

● v versus S curves for glycogen phosphorylase. (a) The sigmoid response of glycogen phosphorylase to the concentration of the substrate phosphate (Pi) shows strong positive cooperativity. (b) ATP is a feedback inhibitor that affects the affinity of glycogen phosphorylase for its substrates but does not affect Vmax. (Glucose-6-P shows similar effects on glycogen phosphorylase.) (c) AMP is a positive heterotropic effector for glycogen phosphorylase. It binds at the same site as ATP. AMP and ATP are competitive. Like ATP, AMP affects the affinity of glycogen phosphorylase for its substrates, but does not affect Vmax.

FIGURE 15.17 ● The mechanism of covalent modification and allosteric regulation of glycogen phosphorylase. The T states are blue and the R states blue-green.

energy (ATP) should be produced. Reciprocal changes in the cellular concentrations of ATP and AMP and their competition for binding to the same site (the allosteric site) on glycogen phosphorylase, with opposite effects, allow these two nucleotides to exert rapid and reversible control over glycogen phosphorylase activity. Such reciprocal regulation ensures that the production of energy (ATP) is commensurate with cellular needs.

To summarize, muscle glycogen phosphorylase is allosterically activated by AMP and inhibited by ATP and glucose-6-P; caffeine can also act as an allosteric inhibitor (Figure 15.17). When ATP and glucose-6-P are abundant, glycogen breakdown is inhibited. When cellular energy reserves are low (i.e., high [AMP] and low [ATP] and [G-6-P]), glycogen catabolism is stimulated.

Glycogen phosphorylase conforms to the Monod–Wyman–Changeux model of allosteric transitions, with the active form of the enzyme designated the R state and the inactive form denoted as the T state (Figure 15.17). Thus, AMP promotes the conversion to the active R state, whereas ATP, glucose-6-P, and caffeine favor conversion to the inactive T state.

X-ray diffraction studies of glycogen phosphorylase in the presence of allosteric effectors have revealed the molecular basis for the T 34 R conversion. Although the structure of the central core of the phosphorylase subunits is identical in the T and R states, a significant change occurs at the subunit interface between the T and R states. This conformation change at the subunit interface is linked to a structural change at the active site that is important for catalysis. In the T state, the negatively charged carboxyl group of Asp283 faces the active site, so that binding of the anionic substrate phosphate is unfavorable. In the conversion to the R state, Asp283 is displaced from the active site and replaced by Arg569. The exchange of negatively charged aspartate for positively charged arginine at the active site provides a favorable binding site for phosphate. These allosteric controls serve as a mechanism for adjusting the activity of glycogen phosphorylase to meet normal metabolic demands. However, in crisis situations in which abundant energy (ATP) is needed imme-

Catalytic site'

Glycogen storage site'

α 2'

AMP' Ser14–P'

Cap

GP a

Cap'

Ser14–P

AMP

α 2

GP b

Glycogen storage site

FIGURE 15.18 ● In this diagram of the glycogen phosphorylase dimer, the phosphorylation site (Ser14) and the allosteric (AMP) site face the viewer. Access to the catalytic site is from the opposite side of the protein. The diagram shows the major conformational change that occurs in the N-terminal residues upon phosphorylation of Ser14. The solid black line shows the conformation of residues 10 to 23 in the b, or unphosphorylated, form of glycogen phosphorylase. The conformational change in the location of residues 10 to 23 upon phosphorylation of Ser14 to give the a (phosphorylated) form of glycogen phosphorylase is shown in yellow. Note that these residues move from intrasubunit contacts into intersubunit contacts at the subunit interface. (Sites on the two respective subunits are denoted, with those of the upper subunit designated by primes ( ).)

(Adapted from Johnson, L. N., and Barford, D., 1993. The effects of phosphorylation on the structure and function of proteins. Annual Review of Biophysics and Biomolecular Structure 22:199–232.)

● The hormone-activated enzymatic cascade that leads to activation of glycogen phosphorylase.

ATP

FIGURE 15.20 ● The adenylyl cyclase reaction yields 3 ,5 -cyclic AMP and pyrophosphate. The reaction is driven forward by subsequent hydrolysis of pyrophosphate by the enzyme inorganic pyrophosphatase.

FIGURE 15.22 ● O2-binding curves for hemoglobin and myoglobin.

Ancient life forms evolved in the absence of oxygen and were capable only of anaerobic metabolism. As the earth’s atmosphere changed over time, so too did living things. Indeed, the production of O2 by photosynthesis was a major factor in altering the atmosphere. Evolution to an oxygen-based metabolism was highly beneficial. Aerobic metabolism of sugars, for example, yields far more energy than corresponding anaerobic processes. Two important oxygenbinding proteins appeared in the course of evolution so that aerobic metabolic processes were no longer limited by the solubility of O2 in water. These proteins are represented in animals as hemoglobin (Hb) in blood and myoglobin (Mb) in muscle. Because hemoglobin and myoglobin are two of the most-stud- ied proteins in nature, they have become paradigms of protein structure and function. Moreover, hemoglobin is a model for protein quaternary structure and allosteric function. The binding of O2 by hemoglobin, and its modulation by effectors such as protons, CO2, and 2,3-bisphosphoglycerate, depend on interactions between subunits in the Hb tetramer. Subunit–subunit interactions in Hb reveal much about the functional significance of quaternary associations and allosteric regulation.

The Comparative Biochemistry of Myoglobin and Hemoglobin

A comparison of the properties of hemoglobin and myoglobin offers insights into allosteric phenomena, even though these proteins are not enzymes. Hemoglobin displays sigmoid-shaped O2-binding curves (Figure 15.22). The unusual shape of these curves was once a great enigma in biochemistry. Such curves closely resemble allosteric enzyme:substrate saturation graphs (see Figure 15.8). In contrast, myoglobin’s interaction with oxygen obeys classical Michaelis–Menten-type substrate saturation behavior.

Before examining myoglobin and hemoglobin in detail, let us first encapsulate the lesson: Myoglobin is a compact globular protein composed of a single polypeptide chain 153 amino acids in length; its molecular mass is 17.2 kD (Figure 15.23). It contains heme, a porphyrin ring system complexing an iron ion, as its prosthetic group (see Figure 5.15). Oxygen binds to Mb via its heme. Hemoglobin (Hb) is also a compact globular protein, but Hb is a tetramer. It consists of four polypeptide chains, each of which is very similar structurally

Partial pressure of oxygen (pO2, mm Hg)