A. Conformational Changes in Allosteric Enzymes
Allosteric activators and inhibitors (allosteric effectors) are compounds that bind to the allosteric site (a site separate from the catalytic site) and cause a conforma- tional change that affects the affi nity of the enzyme for the substrate. Usually, an allosteric enzyme has multiple interacting subunits that can exist in active and inac- tive conformations and the allosteric effector promotes or hinders conversion from one conformation to another.
1. COOPERATIVITY IN SUBSTRATE BINDING TO ALLOSTERIC ENZYMES
Allosteric enzymes usually contain two or more subunits and exhibit positive co- operativity; the binding of substrate to one subunit facilitates the binding of sub- strate to another subunit. The fi rst substrate molecule has diffi culty in binding to the enzyme because all of the subunits are in the conformation with a low affi nity for substrate (the taut “T” conformation). The fi rst substrate molecule to bind changes its own subunit and at least one adjacent subunit to the high affi nity conformation (the relaxed “R” state). In the example of the tetramer hemoglobin discussed in Chapter 6, the change in one subunit facilitated changes in all four subunits, and the molecule generally changed to the new conformation in a concerted fashion.
However, most allosteric enzymes follow a more stepwise (sequential) progression through intermediate stages.
2. ALLOSTERIC ACTIVATORS AND INHIBITORS
Allosteric enzymes bind activators at the allosteric site, a site physically separate from the catalytic site. The binding of an allosteric activator changes the conformation of the catalytic site in a way that increases the affi nity of the enzyme for the substrate.
In general, activators of allosteric enzymes bind more tightly to the high affi n- ity R state of the enzyme than the T state (i.e., the allosteric site is open only in the R enzyme) (Fig. 7.7). Thus, the activators increase the amount of enzyme in the active state, thereby facilitating substrate binding in their own and other subunits.
In contrast, allosteric inhibitors bind more tightly to the T state, so either substrate concentration or activator concentration must be increased to overcome the effects of the allosteric inhibitor.
In the absence of activator, a plot of velocity versus substrate concentration for an allosteric enzyme usually results in a sigmoid or S-shaped curve (rather than the rectangular hyperbola of Michaelis-Menten enzymes; allosteric enzymes do not obey Michaelis-Menten kinetics) as the successive binding of substrate molecules activates additional subunits (see Fig. 7.7). In plots of velocity versus substrate concentration, the effect of an allosteric activator generally makes the sigmoidal S-shaped curve more like the rectangular hyperbola, with a substantial decrease in the S0.5 (Km ) of the en- zyme, because the activator changes all of the subunits to the high affi nity state. These allosteric effectors alter the Km but not the Vmax of the enzyme. An allosteric inhibitor makes it more diffi cult for substrate or activators to convert the subunits to the most active conformation, and therefore, inhibitors generally shift the curve to the right, either increasing the S0.5 alone or increasing it together with a decrease in the Vmax.
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CHAPTER 7 ■ REGULATION OF ENZYMES 103
C. ALLOSTERIC ENZYMES IN METABOLIC PATHWAYS
Regulation of enzymes by allosteric effectors provides several advantages over other methods of regulation. Allosteric inhibitors usually have a much stronger effect on enzyme velocity than competitive, noncompetitive, and uncompetitive inhibitors in the active catalytic site. Because allosteric effectors do not occupy the catalytic site, they may function as activators. Thus, allosteric enzymes are not limited to regula- tion through inhibition. Furthermore, the allosteric effector need not bear any re- semblance to substrate or product of the enzyme. Finally, the effect of an allosteric effector is rapid, occurring as soon as its concentration changes in the cell. These features of allosteric enzymes are often essential for feedback regulation of meta- bolic pathways by end products of the pathway or by signal molecules that coordi- nate multiple pathways.
B. Conformational Changes from Covalent Modifi cation
1. PHOSPHORYLATION
The activity of many enzymes is regulated through phosphorylation by a protein kinase or dephosphorylation by a protein phosphatase (Fig. 7.8). Serine/threonine protein kinases transfer a phosphate from ATP to the hydroxyl group of a specifi c FIG. 7.7. Activators and inhibitors of an allosteric enzyme (simplifi ed model). This enzyme has two identical subunits, each containing three binding sites: one for the substrate (s), one for the allosteric activator (green triangle), and one for the allosteric inhibitor (two-pronged red shape). The enzyme has two conformations, a relaxed active conformation (R) and an inactive conformation (T). The activator binds only to its activator site when the enzyme is in the R confi guration. The inhibitor-binding site is open only when the enzyme is in the T state. A plot of velocity (vi/Vmax) versus substrate concentration reveals that binding of the substrate at its binding site stabilizes the active conformation so that the second substrate binds more readily, resulting in an S (sigmoidal)-shaped curve. The graph of vi/Vmax be- comes hyperbolic in the presence of activator (which stabilizes the high affi nity R form) and more sigmoidal with a higher S0.5 in the presence of inhibitor (which stabilizes the low affi nity form).
S S
S S
A model of an allosteric enzyme
R T
Substrate
Substrate Activator Inhibitor
Activator
S S
S S
S S S
S
Vmax vi
S0.5
[S]
0 1.0 2.0
0.5 1.0
No activator or inhibitor
0
+ Activator
+ Inhibitor
S0.5
CH2OH
Protein with serine side chain
CH2
HO P O O– O
ADP
+ O P O–
O– O
ADP ATP
Phosphorylated protein Protein kinase H2O
–O OH
O– O
Protein phosphatase
P
FIG. 7.8. Protein kinases and protein phos- phatases.
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serine (and sometimes threonine) on the target enzyme; tyrosine kinases transfer a phosphate to the hydroxyl group of a specifi c tyrosine residue. Phosphate is a bulky, negatively charged residue that interacts with other nearby amino acid resi- dues of the protein to create a conformational change at the catalytic site. The con- formational change is caused by alterations in ionic interactions and/or hydrogen bond patterns due to the presence of the phosphate group. The conformational change makes certain enzymes more active and other enzymes less active. The ef- fect is reversed by a specifi c protein phosphatase that removes the phosphate by hydrolysis.
2. OTHER COVALENT MODIFICATIONS
A number of proteins are covalently modifi ed by the addition of groups such as acetyl, ADP-ribose, or lipid moieties (see Chapter 4). These modifi cations may directly activate or inhibit the enzyme. However, they may also modify the abil- ity of the enzyme to interact with other proteins or to reach its correct location in the cell.
C. Conformational Changes Regulated by Protein-Protein Interactions
Changes in the conformation of the active site can also be regulated by direct protein- protein interaction. This type of regulation is illustrated by protein kinase A, calcium (Ca2)-binding proteins, and small (monomeric) G proteins.
1. PROTEIN KINASE A
Some protein kinases are tightly bound to a single protein and regulate only the protein to which they are tightly bound. However, other protein kinases and protein phosphatases will simultaneously regulate a number of rate-limiting enzymes in a cell to achieve a coordinated response. For example, protein kinase A, a serine/
threonine protein kinase, phosphorylates a number of enzymes that regulate differ- ent metabolic pathways.
Protein kinase A provides a means for hormones to control metabolic pathways.
Epinephrine (adrenaline) and many other hormones increase the intracellular con- centration of the allosteric regulator 3,5-cyclic AMP (cAMP), which is referred to as a hormonal second messenger (Fig. 7.9A). cAMP binds to regulatory subunits of protein kinase A, which dissociate and release the activated catalytic subunits (see Fig. 7.9B). Dissociation of inhibitory regulatory subunits is a common theme in enzyme regulation. The active catalytic subunits phosphorylate proteins at serine and threonine residues.
2. THE CALCIUM-CALMODULIN FAMILY OF MODULATOR PROTEINS
Modulator proteins bind to other proteins and regulate their activity by causing a conformational change at the catalytic site or by blocking the catalytic site (steric hindrance). They are protein allosteric effectors that can either activate of inhibit the enzyme or protein to which they bind.
Ca2-calmodulin is an example of a dissociable modulator protein that binds to several different proteins and regulates their function in either a positive or nega- tive manner. It also exists in the cytosol and functions as a Ca2-binding protein (Fig. 7.10). The center of the symmetric molecule is a hinge region that bends as Ca2-calmodulin folds over the protein it is regulating.
3. G PROTEINS
The masters of regulation through reversible protein association in the cell are the monomeric G proteins, small single subunit proteins that bind and hydrolyze gua- nosine triphosphate (GTP). GTP is a purine nucleotide which, like ATP, contains A
B
C C
C C R
R
R R
+ cAMP binding
Active protein kinase A Inactive protein kinase A
O
–O CH2
NH2
O
O P
O OH
H
H H H
N N N
C N H
CH C C C
FIG. 7.9. A. Structure of cAMP (3,5-cyclic AMP). The phosphate group is attached to hydroxyl groups on both the third (3) and fi fth (5) carbons of ribose, forming a cyclic structure. B. Protein kinase A. When the regu- latory subunits (R) of protein kinase A bind the allosteric activator, cAMP, they dissociate from the enzyme, thereby releasing active cata- lytic subunits (C).
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CHAPTER 7 ■ REGULATION OF ENZYMES 105
high-energy phosphoanhydride bonds that release energy when hydrolyzed. When G proteins bind GTP, their conformation changes so that they can bind to a target protein, which is then either activated or inhibited in carrying out its function (Fig. 7.11, circle 1).
G proteins are said to possess an internal clock because they are GTPases that slowly hydrolyze their own bound GTP to GDP and phosphate. As they hydrolyze GTP, their conformation changes and the complex they have formed with the target protein disassembles (see Fig. 7.11, circle 2). The bound GDP on the inactive G pro- tein is eventually replaced by GTP, and the process can begin again (see Fig. 7.11, circle 3).
The activity of many G proteins is regulated by accessory proteins (GAPs, GEFs, and GDIs), which may, in turn, be regulated by allosteric effectors. GAPs (GTPase activating proteins) increase the rate of GTP hydrolysis by the G protein and, therefore, the rate of dissociation of the G protein–target protein complex (see Fig. 7.11, circle 2). When a GEF protein (guanine nucleotide exchange fac- tor) binds to a G protein, it increases the rate of GTP exchange for a bound GDP and therefore activates the G protein (see Fig. 7.11, circle 3). GDI proteins (GDP dissociation inhibitor) bind to the GDP–G protein complex and inhibit dissociation of GDP, thereby keeping the G protein inactive. G proteins are discussed in more detail in Chapter 8.
D. Proteolytic Cleavage
Although many enzymes undergo some cleavage during synthesis, others enter ly- sosomes or secretory vesicles or are secreted as proenzymes, which are precursor proteins that must undergo proteolytic cleavage to become fully functional. Unlike most other forms of regulation, proteolytic cleavage is irreversible.
The precursor proteins of proteases (enzymes that cleave specifi c peptide bonds) are called zymogens. To denote the inactive zymogen form of an enzyme, the
Flexible region between domains
FIG. 7.10. Ca2-calmodulin has four binding sites for Ca2 (shown in green). Each Ca2 forms a multiligand coordination sphere by simultaneously binding several amino acid resi- dues on calmodulin. Thus, calmodulin can create large conformational changes in proteins to which it is bound when Ca2 binds. Calmodulin has a fl exible region in the middle connecting the two domains.
GTP Association Inactive
target protein
Active G-protein
Active G-protein
GTP hydrolysis and dissociation Activated
target protein
Pi
Nucleotide exchange Inactive
target protein
Inactive G-protein
GDP GTP
GDP 1
2
3
FIG. 7.11. Monomeric G proteins. Step 1:
When GTP is bound, the conformation of the G protein allows it to bind target proteins, which are then activated (as shown) or inhibited.
Step 2: The G protein hydrolyzes a phosphate from GTP to form guanosine diphosphate (GDP), which changes the G-protein confor- mation and causes it to dissociate from the target protein. Step 3: GDP is exchanged for GTP, which reactivates the G protein.
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name is modifi ed by addition of the suffi x “-ogen” or the prefi x “pro-.” The synthe- sis of zymogens as inactive precursors prevents them from cleaving proteins pre- maturely at their sites of synthesis or secretion. Chymotrypsinogen, for example, is stored in vesicles within pancreatic cells until secreted into ducts leading to the intestinal lumen. In the digestive tract, chymotrypsinogen is converted to chy- motrypsin by the proteolytic enzyme trypsin, which cleaves off a small peptide from the N-terminal region (and two internal peptides). This cleavage activates chymotrypsin by causing a conformational change in the spacing of amino acid residues around the binding site for the denatured protein substrate and around the catalytic site.
An excellent example of zymogen activation is in the mechanism of blood clot- ting. It is critical for individuals to maintain a constant blood volume (hemostasis), so injuries to the integrity of blood vessels must be repaired and closed before a signifi cant amount of blood is lost. When damage to the circulatory system is found, a cascade of zymogen activation is initiated in order to form a clot (consisting of the protein fi brin), which seals the damaged area. Fibrin is initially synthesized as an inactive precursor, fi brinogen, which is converted to fi brin by the serine protease thrombin.
Thrombin activation is mediated by the complex interaction which comprises the blood coagulation cascade. This cascade (Fig. 7.12) consists primarily of pro- teins that serve as enzymes or cofactors, which function to accelerate thrombin
Intrinsic pathway Extrinsic pathway
XI
IX
XIa
VIII VIIIa
V Va
XIII XIIIa
IXa
VIIa VII
Tissue factor (III) Trauma
X X
Xa
II
Prothrombin IIa Thrombin
I Fibrinogen
Ia Fibrin aggregate
(soft clot)
Cross-linked clot (hard clot)
PL, Ca PL, Ca
PL, Ca PL, Ca
PL, Ca
FIG. 7.12. The blood coagulation cascade. Activation of clot formation occurs through in- terlocking pathways, termed the intrinsic and extrinsic pathways. The intrinsic pathway is activated when factor XI is converted to factor XIa (the active form) by thrombin. The extrin- sic pathway (external damage, such as a cut) is activated by tissue factor. The reactions desig- nated by “PL, Ca” are occurring via cofactors bound to phospholipids (PL) on the platelet and blood vessel endothelial cell surface in a Ca2-coordination complex. Factors XIa, IXa, VIIa, Xa, and thrombin are serine proteases. Note the positive feedback regulation of thrombin on the activation of proteases earlier in the cascade sequence (indicated by the dashed lines).
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CHAPTER 7 ■ REGULATION OF ENZYMES 107
formation and localize it at the site of injury. These proteins are listed in Table 7.1.
All of these proteins are present in the plasma as proproteins (zymogens). These precursor proteins are activated by cleavage of the polypeptide chain at one or more sites. The key to successful and appropriate clot (thrombus) formation is the regula- tion of the proteases, which activate these zymogens.
The proenzymes (factors VII, IX, X, XI, and prothrombin) are serine proteases that, when activated by cleavage, cleave the next proenzyme in the cascade. Because of the sequential activation, great acceleration and amplifi cation of the response is achieved.
That cleavage and activation have occurred is indicated by the addition of an “a” to the name of the proenzyme (e.g., factor IX is cleaved to form the active factor IXa).
The cofactor proteins (tissue factor, factors V and VIII) serve as binding sites for other factors. Tissue factor is not related structurally to the other blood coagulation cofactors and is an integral membrane protein that does not require cleavage for ac- tive function. Factors V and VIII serve as pro-cofactors, which, when activated by cleavage, function as binding sites for other factors.
Two additional proteins that are considered part of the blood coagulation cas- cade, protein S and protein C, are regulatory proteins. Only protein C is regulated by proteolytic cleavage, and when activated, is itself a serine protease. These proteins are critical to stopping clot formation when the damage has been repaired. A more detailed discussion of blood clotting can be found online in Section A7.1.