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

FIGURE 15.3 ● Proinsulin is an 86-residue precursor to insulin (the sequence shown here is human proinsulin). Proteolytic removal of residues 31 to 65 yields insulin. Residues 1 through 30 (the B chain) remain linked to residues 66 through 87 (the A chain) by a pair of interchain disulfide bridges.

Zymogens

Most proteins become fully active as their synthesis is completed and they spontaneously fold into their native, three-dimensional conformations. Some proteins, however, are synthesized as inactive precursors, called zymogens or proenzymes, that only acquire full activity upon specific proteolytic cleavage of one or several of their peptide bonds. Unlike allosteric regulation or covalent modification, zymogen activation by specific proteolysis is an irreversible process. Activation of enzymes and other physiologically important proteins by specific proteolysis is a strategy frequently exploited by biological systems to switch on processes at the appropriate time and place, as the following examples illustrate.

INSULIN. Some protein hormones are synthesized in the form of inactive precursor molecules, from which the active hormone is derived by proteolysis. For instance, insulin, an important metabolic regulator, is generated by proteolytic excision of a specific peptide from proinsulin (Figure 15.3).

PROTEOLYTIC ENZYMES OF THE DIGESTIVE TRACT. Enzymes of the digestive tract that serve to hydrolyze dietary proteins are synthesized in the stomach and pancreas as zymogens (Table 15.1). Only upon proteolytic activation are these enzymes able to form a catalytically active substrate-binding site. The activation of chymotrypsinogen is an interesting example (Figure 15.4). Chymotrypsinogen is a 245-residue polypeptide chain cross-linked by five disulfide bonds. Chymotrypsinogen is converted to an enzymatically active form called -chy- motrypsin when trypsin cleaves the peptide bond joining Arg15 and Ile16. The

enzymatically active -chymotrypsin acts upon other -chymotrypsin molecules, excising two dipeptides, Ser14-Arg15 and Thr147-Asn148. The end product

of this processing pathway is the mature protease -chymotrypsin, in which the three peptide chains, A (residues 1 through 13), B (residues 16 through 146), and C (residues 149 through 245), remain together because they are linked by two disulfide bonds, one from A to B, and one from B to C. It is interesting to note that the transformation of inactive chymotrypsinogen to active -chy- motrypsin requires the cleavage of just one particular peptide bond.

BLOOD CLOTTING. The formation of blood clots is the result of a series of zymogen activations (Figure 15.5). The amplification achieved by this cascade of enzymatic activations allows blood clotting to occur rapidly in response to injury. Seven of the clotting factors in their active form are serine proteases:

Table 15.1

Pancreatic and Gastric Zymogens

● The cascade of activation steps leading to blood clotting. The intrinsic and extrinsic pathways converge at Factor X, and the final common pathway involves the activation of thrombin and its conversion of fibrinogen into fibrin, which aggregates into ordered filamentous arrays that become crosslinked to form the clot.

Protein kinases are converter enzymes that catalyze the ATPdependent phosphorylation of serine, threonine, and/or tyrosine hydroxyl groups in target proteins (table). Phosphorylation introduces a bulky group bearing two negative charges, causing conformational changes that alter the target protein’s function. (Unlike a phosphoryl group, no amino acid side chain can provide two negative charges.) Protein kinases represent a protein superfamily whose members are widely diverse in terms of size, subunit structure, and subcellular localization. Nevertheless, all share a common catalytic mechanism based on a conserved catalytic core/kinase domain of approximately 260 amino acid residues (see figure). Protein kinases are classified as Ser/Thrand/or Tyr-specific and are subclassified in terms of the allosteric activators they require and the consensus amino acid sequence within the target protein that is recognized by the kinase. For example, cAMP-dependent protein kinase (PKA) phosphorylates proteins having Ser or Thr residues within an R(R/K)X(S*/T*) target consensus sequence (* denotes the residue that becomes phosphorylated). That is, PKA phosphorylates Ser or Thr residues that occur in an Arg-(Arg or Lys)-(any amino acid)-(Ser or Thr) sequence segment (table).

Targeting of protein kinases to particular consensus sequence elements within proteins creates a means to regulate these kinases by intrasteric control. Intrasteric control occurs when a regulatory subunit (or protein domain) has a pseudosubstrate sequence that mimics the target sequence but lacks a OH-bearing side chain at the right place. For example, the cAMPbinding regulatory subunits of PKA (R subunits in Figure 15.7) possess the pseudosubstrate sequence RRGA*I and this sequence binds to the active site of PKA catalytic subunits, blocking their activity. This pseudosubstrate sequence has an alanine residue where serine occurs in the PKA target sequence; Ala is sterically similar to serine but lacks a phosphorylatable OH-group. When

these PKA regulatory subunits bind cAMP, they undergo a conformational change and dissociate from the catalytic (C) subunits, and the active site of PKA is free to bind and phosphorylate its targets.

The abundance of many protein kinases in cells is an indication of the great importance of protein phosphorylation in cellular regulation. Exactly 113 protein kinase genes have been recognized in yeast, and it is estimated that the human genome encodes more than 1000 different protein kinases. Tyrosine kinases (protein kinases that phosphorylate Tyr residues) occur only in multicellular organisms (yeast has no tyrosine kinases). Tyrosine kinases are components of signaling pathways involved in cell–cell communication (see Chapter 34).

Cyclic AMP-dependent protein kinase is shown complexed with a pseudosubstrate peptide (red). This complex also includes ATP (yellow) and two Mn2 ions (violet) bound at the active site.

White cells

A3B

Brain

Kidney

AB3

Heart

B4

● The isozymes of lactate dehydrogenase (LDH). Active muscle tissue becomes anaerobic and produces pyruvate from glucose via glycolysis (Chapter 19). It needs LDH to regenerate NAD from NADH so glycolysis can continue. The lactate produced is released into the blood. The muscle LDH isozyme (A4) works best in the NAD - regenerating direction. Heart tissue is aerobic and uses lactate as a fuel, converting it to pyruvate via LDH and using the pyruvate to fuel the citric acid cycle to obtain energy. The heart LDH isozyme (B4) is inhibited by excess pyruvate so the fuel won’t be wasted.

General Properties of Regulatory Enzymes

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:

1.Their kinetics do not obey the Michaelis–Menten equation. Their v versus

[S]plots yield sigmoid- or S-shaped curves rather than rectangular hyper-

bolas (Figure 15.8). Such curves suggest a second-order (or higher) relationship between v and [S]; that is, v is proportional to [S]n, where n 1.

A qualitative description of the mechanism responsible for the S-shaped curves is that binding of one S to a protein molecule makes it easier for additional substrate molecules to bind to the same protein molecule. In the jargon of allostery, substrate binding is cooperative.

2.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.

3.Regulatory or allosteric enzymes like enzyme 1 are, in some instances, regulated by activation. That is, whereas some effector molecules such as F exert negative effects on enzyme activity, other effectors show stimulatory, or positive, influences on activity.

4.Allosteric enzymes have an oligomeric organization. They are composed of more than one polypeptide chain (subunit) and have more than one S-bind- ing site per enzyme molecule.

5.The working hypothesis is that, by some means, interaction of an allosteric enzyme with effectors alters the distribution of conformational possibilities or subunit interactions available to the enzyme. That is, the regulatory effects exerted on the enzyme’s activity are achieved by conformational changes occurring in the protein when effector metabolites bind.

In addition to enzymes, noncatalytic proteins may exhibit many of these properties; hemoglobin is the classic example. The allosteric properties of hemoglobin are the subject of a Special Focus beginning on page 480.

15.4 ● Models for the Allosteric Behavior of Proteins

The Symmetry Model of Monod, Wyman, and Changeux

In 1965, Jacques Monod, Jeffries Wyman, and Jean-Pierre Changeux proposed a theoretical model of allosteric transitions based on the observation that allosteric proteins are oligomers. They suggested that allosteric proteins can exist in (at least) two conformational states, designated R, signifying “relaxed,” and T, or “taut,” and that, in each protein molecule, all of the subunits have the same conformation (either R or T). That is, molecular symmetry is conserved. Molecules of mixed conformation (having subunits of both R and T states) are not allowed by this model.

In the absence of ligand, the two states of the allosteric protein are in equilibrium:

R0 34 T0

(Note that the subscript “0” signifies “in the absence of ligand.”) The equilibrium constant is termed L: L T0/R0. L is assumed to be large; that is, the

469

Hyperbolic

Sigmoid

[S]

● Sigmoid v versus [S] plot. The dotted line represents the hyperbolic plot characteristic of normal Michaelis–Menten-type enzyme kinetics.

(a) A dimeric protein can exist in either

of two conformational states at equilibrium.

L

Substrate

R1 bound

FIGURE 15.9 ● Monod–Wyman–Changeux (MWC) model for allosteric transitions. Consider a dimeric protein that can exist in either of two conformational states, R or T. Each subunit in the dimer has a binding site for substrate S and an allosteric effector site, F. The promoters are symmetrically related to one another in the protein, and symmetry is conserved regardless of the conformational state of the protein. The different states of the protein, with or without bound ligand, are linked to one another through the various equilibria. Thus, the relative population of protein molecules in the R or T state is a function of these equilibria and the concentration of the various ligands, substrate (S), and effectors (which bind at FR or FT). As [S] is increased, the T/R equilibrium shifts in favor of an increased proportion of R-conformers in the total population (that is, more protein molecules in the R conformational state).

amount of the protein in the T conformational state is much greater than the amount in the R conformation. Let us suppose that L 104.

The affinities of the two states for substrate, S, are characterized by the respective dissociation constants, KR and KT. The model supposes that KT KR. That is, the affinity of R0 for S is much greater than the affinity of T0 for S. Let us choose the extreme where KR/KT 0 (that is, KT is infinitely greater than KR). In effect, we are picking conditions where S binds only to R. (If KT is infinite, T does not bind S.)

Given these parameters, consider what happens when S is added to a solution of the allosteric protein at conformational equilibrium (Figure 15.9). Although the relative [R0] concentration is small, S will bind “only” to R0, forming R1. This depletes the concentration of R0, perturbing the T0/R0 equilibrium. To restore equilibrium, molecules in the T0 conformation undergo a transition to R0. This shift renders more R0 available to bind S, yielding R1, diminishing [R0], perturbing the T0/R0 equilibrium, and so on. Thus, these linked equilibria (Figure 15.9) are such that S-binding by the R0 state of the allosteric protein perturbs the T0/R0 equilibrium with the result that S-bind- ing drives the conformational transition, T0 n R0.

In just this simple system, cooperativity is achieved because each subunit has a binding site for S, and thus, each protein molecule has more than one binding site for S. Therefore, the increase in the population of R conformers gives a progressive increase in the number of sites available for S. The extent of cooperativity depends on the relative T0/R0 ratio and the relative affinities of R and T for S. If L is large (that is, the equilibrium lies strongly in favor of T0) and if KT KR, as in the example we have chosen, cooperativity is great (Figure 15.10). Ligands such as S here that bind in a cooperative manner, so that binding of one equivalent enhances the binding of additional equivalents of S to the same protein molecule, are termed positive homotropic effectors. (The prefix “homo” indicates that the ligand influences the binding of like molecules.)

a tetramer (n 4) in terms of Y, the saturation function, versus [S]. Y is defined as [lig- and-binding sites that are occupied by ligand]/[total ligand-binding sites]. (a) A plot of Y as a function of [S], at various L values. (b) Y as a function of [S], at different c, where

c KR/KT. (When c 0, KT is infinite.) (Adapted from Monod, J., Wyman, J., and Changeux, J.-P., 1965. On the nature of allosteric transitions: A plausible model. Journal of Molecular Biology 12:92.)