For example, protein kinases are enzymes that act in covalent modification by attaching a phosphoryl moiety to target proteins Figure 15.1.. Enzyme OH Protein phosphatase Protein kinase E
Trang 1of enzyme synthesis, are important mechanisms for the regulation of metabolism.
By controlling the amount of an enzyme that is present at any moment, cells can
ei-ther activate or terminate various metabolic routes Genetic controls over enzyme
levels have a response time ranging from minutes in rapidly dividing bacteria to
hours (or longer) in higher eukaryotes Once synthesized, the enzyme may also be
degraded, either through normal turnover of the protein or through specific decay
mechanisms that target the enzyme for destruction These mechanisms are
dis-cussed in detail in Chapter 31
Enzyme Activity Can Be Regulated Allosterically
Enzymatic activity can also be activated or inhibited through noncovalent interaction
of the enzyme with small molecules (metabolites) other than the substrate This
form of control is termed allosteric regulation, because the activator or inhibitor
binds to the enzyme at a site other than (allo means “other”) the active site
Further-more, such allosteric regulators, or effector molecules, are often quite different
ster-ically from the substrate Because this form of regulation results simply from
re-versible binding of regulatory ligands to the enzyme, the cellular response time can
be virtually instantaneous
Enzyme Activity Can Be Regulated Through Covalent Modification
Enzymes can be regulated by covalent modification, the reversible covalent attachment
of a chemical group Enzymes susceptible to such regulation are called
interconvert-ible enzymes, because they can be reversibly converted between two forms Thus, a
fully active enzyme can be converted into an inactive form simply by the covalent
at-tachment of a functional group For example, protein kinases are enzymes that act in
covalent modification by attaching a phosphoryl moiety to target proteins (Figure
15.1) Protein kinases catalyze the ATP-dependent phosphorylation of OOH groups
on Ser, Thr, or Tyr side chains Removal of the phosphate group by a phosphoprotein
phosphatasereturns the enzyme to its original state In contrast to the example in the
figure, some enzymes exist in an inactive state unless specifically converted into the
active form through covalent addition of a functional group Covalent modification
reactions are catalyzed by special converter enzymes, which are themselves subject to
metabolic regulation (Protein kinases are one class of converter enzymes.) Although
covalent modification represents a stable alteration of the enzyme, a different
con-verter enzyme operates to remove the modification, so when the conditions that
fa-vored modification of the enzyme are no longer present, the process can be reversed,
restoring the enzyme to its unmodified state Because covalent modification events are
catalyzed by enzymes, they occur very quickly, with response times of seconds or even
less for significant changes in metabolic activity
Regulation of Enzyme Activity Also Can Be Accomplished
in Other Ways
Enzyme regulation is an important matter to cells, and evolution has provided a
vari-ety of additional options, including zymogens, isozymes, and modulator proteins We
will discuss these options first and then return to the major topics of this chapter—
enzyme regulation through allosteric mechanisms and covalent modification
Enzyme OH
Protein phosphatase
Protein kinase
Enzyme O P O–
O–
Catalytically inactive, covalently modified form
Catalytically
active form
O
H O P
FIGURE 15.1 Enzyme regulation by reversible covalent modification.
Trang 2454 Chapter 15 Enzyme Regulation
Zymogens Are Inactive Precursors of Enzymes
Most proteins become fully active as their synthesis is completed and they sponta-neously fold into their native, three-dimensional conformations Some proteins,
how-ever, are synthesized as inactive precursors, called zymogens or proenzymes, that
ac-quire full activity only upon specific proteolytic cleavage of one or several of their peptide bonds Unlike allosteric regulation or covalent modification, zymogen activa-tion by specific proteolysis is an irreversible process Activaactiva-tion of enzymes and other physiologically important proteins by specific proteolysis is a strategy frequently ex-ploited 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 15.2).
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
chymotrypsino-gen is an interesting example (Figure 15.3) Chymotrypsinochymotrypsino-gen is a 245-residue
polypeptide chain crosslinked by five disulfide bonds Chymotrypsinogen is con-verted to an enzymatically active form called -chymotrypsin when trypsin cleaves
the peptide bond joining Arg15and Ile16 The enzymatically active -chymotrypsin
acts upon other -chymotrypsin molecules, excising two dipeptides: Ser14–Arg15
Proinsulin
Val
Phe
NH3
1
Asn
Gln
His
Leu
Cys
Gly
Ser
His
Leu
Val
Glu
Ala
Leu
Tyr
Leu
Val
Cys
Gly
Glu
Arg
Gly
Phe
Phe
Tyr
Thr
Pro
Thr
10
20
30
Arg
Arg GluAla
Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gly Gly Leu Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly
Leu
Gln Ser Gln
Lys
Arg
Gly
40 50
60 65
Ile
Val
Glu
Gln
Cys
Cys
Thr
Ser
Ile
Cys
Ser
Leu
Tyr
Gln
Leu
Glu
Asn
Tyr
Cys
Asn
1
10
21
COO–
S
S
S
S
S
S
Connecting peptide
Insulin
Val Phe
NH3 1
Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr
Lys Thr
10
20
30
Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu Glu Asn Tyr Cys Asn
1
10
21 COO–
S
S
S
S
S
S Pro Lys
FIGURE 15.2 Proinsulin is an 86-residue precursor to
in-sulin (the sequence shown here is human proinin-sulin).
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
inter-chain disulfide bridges.
245 1
Chymotrypsinogen (inactive zymogen)
Cleavage at Arg 15
by trypsin
1
-Chymotrypsin (active enzyme)
Self-digestion at Leu13, Tyr 146 , and Asn 148 by
-chymotrypsin
1
-Chymotrypsin (active enzyme)
Arg
13 13
245
148 147 15
14
148 147 15
14
148 147
14 15
ANIMATED FIGURE 15.3 The
proteo-lytic activation of chymotrypsinogen See this figure
animated at www.cengage.com/login.
TABLE 15.1 Pancreatic and Gastric Zymogens
Trang 3and Thr147–Asn148 The end product of this processing pathway is the mature
pro-tease ␣-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
Blood Clotting The formation of blood clots is the result of a series of zymogen
activations (Figure 15.4) 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: kallikrein, XII a , XI a , IX a ,
VII a , X a , and thrombin Two routes to blood clot formation exist The intrinsic
pathwayis instigated when the blood comes into physical contact with abnormal
surfaces caused by injury; the extrinsic pathway is initiated by factors released from
injured tissues The pathways merge at factor X and culminate in clot formation
Thrombin excises peptides rich in negative charge from fibrinogen, converting it to
fibrin,a molecule with a different surface charge distribution Fibrin readily
aggre-gates into ordered fibrous arrays that are subsequently stabilized by covalent
crosslinks Thrombin specifically cleaves Arg–Gly peptide bonds and is homologous
to trypsin, which is also a serine protease (recall that trypsin acts only at Arg and Lys
residues)
Isozymes Are Enzymes with Slightly Different Subunits
A number of enzymes exist in more than one quaternary form, differing in their
relative proportions of structurally equivalent but catalytically distinct polypeptide
subunits A classic example is mammalian lactate dehydrogenase (LDH), which
exists as five different isozymes, depending on the tetrameric association of two
different subunits, A and B: A , A B, A B , AB, and B (Figure 15.5) The kinetic
Intrinsic pathway
Damaged tissue surface
Kininogen
Kallikrein
VIIIa
Tissue
Trauma
Extrinsic pathway
Va
II (Prothrombin)
IIa (Thrombin) I
(Fibrinogen)
Ia (Fibrin) XIIIa Crosslinked fibrin clot
Final common pathway
FIGURE 15.4 The cascade of activation steps leading to blood clotting The intrinsic and extrinsic pathways con-verge at factor X, and the final common pathway involves the activation of thrombin and its conversion of fibrino-gen into fibrin, which aggregates into ordered filamen-tous arrays that become crosslinked to form the clot.
Trang 4456 Chapter 15 Enzyme Regulation
properties of the various LDH isozymes differ in terms of their relative affinities for the various substrates and their sensitivity to inhibition by product Different tissues express different isozyme forms, as appropriate to their particular meta-bolic needs By regulating the relative amounts of A and B subunits they synthe-size, the cells of various tissues control which isozymic forms are likely to assemble and thus which kinetic parameters prevail
of Allosteric Regulation?
Allosteric regulation acts to modulate enzymes situated at key steps in metabolic pathways Consider as an illustration the following pathway, where A is the precur-sor for formation of an end product, F, in a sequence of five enzyme-catalyzed reactions:
enz 1 enz 2 enz 3 enz 4 enz 5
A⎯⎯→ B ⎯⎯→ C ⎯⎯→ D ⎯⎯→ E ⎯⎯→ F
In this scheme, F symbolizes an essential metabolite, such as an amino acid or a
nu-cleotide In such systems, F, the essential end product, inhibits enzyme 1, the first step
in the pathway Therefore, when sufficient F is synthesized, it blocks further
synthe-sis of itself This phenomenon is called feedback inhibition or feedback regulation.
Regulatory Enzymes Have Certain Exceptional Properties
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
cer-tain 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 hyperbolas (Figure
15.6) 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 pro-tein 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) The five isomers of lactate dehydrogenase
A4
A3B
A2B2
AB3
B4
(b) A4 A3B A2B2 AB3 B4
Liver
Muscle White cells Brain
Red cells
Kidney
Heart
ACTIVE FIGURE 15.5 The isozymes of
lactate dehydrogenase (LDH) Active muscle tissue
becomes anaerobic and produces pyruvate from glucose
via glycolysis (see Chapter 18) It needs LDH to regenerate
NADfrom NADH so that glycolysis can continue The
lactate produced is released into the blood The muscle
LDH isozyme (A 4 ) 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 (B 4 ) is inhibited by excess pyruvate so that
the fuel won’t be wasted Test yourself on the
con-cepts in this figure at www.cengage.com/login.
v
[S]
Hyperbolic
Sigmoid
Vmax
FIGURE 15.6 Sigmoid v versus [S] plot The dotted line
represents the hyperbolic plot characteristic of normal
Michaelis–Menten-type enzyme kinetics.
Trang 5a 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,
influ-ences on activity
4 Allosteric enzymes typically have an oligomeric organization They are
com-posed of more than one polypeptide chain (subunit), and each subunit has a
binding site for substrate, as well as a distinct binding site for allosteric
effec-tors Thus, allosteric enzymes typically have more than one S-binding site and
more than one effector-binding site per enzyme molecule
5 The working hypothesis is that, by some means, interaction of an allosteric
en-zyme with effectors alters the distribution of conformational possibilities or
sub-unit 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
prop-erties; hemoglobin is the classic example The allosteric properties of
hemoglo-bin are the subject of a Special Focus at the end of this chapter
by Conformational Changes in Proteins?
The Symmetry Model for Allosteric Regulation Is Based
on Two Conformational States for a Protein
Various models have been proposed to account for the behavior of allosteric
pro-teins All of them note that proteins can exist in different conformational states
Models usually propose a small number of conformations (two or, at most, three)
for a given protein For example, the model for allosteric behavior of Jacques
Monod, Jeffries Wyman, and Jean-Pierre Changeux (the MWC model) proposes two
conformational states for an allosteric protein: the R (relaxed) state and the T
(taut) state The MWC model is sometimes referred to as the symmetry model
be-cause all subunits in an oligomer are assumed to have the same conformation,
whether it is R or T R-state and T-state protein molecules are in equilibrium, with
the T conformation greatly favored over the R ([T] [R]), under conditions in
which no ligands are present This model further suggests that substrate and
al-losteric activators (positive effectors) bind only to the R state and alal-losteric
in-hibitors (negative effectors) bind only to the T state Figure 15.7 illustrates such a
model for a dimeric protein, each monomer of which has a substrate-binding site
and an effector-binding site Because substrate (S) binds only to the R state, S
bind-ing perturbs the R st T equilibrium in favor of more R-state conformers and thus
more S binding That is, S binding is cooperative The concentration of ligand
giv-ing half-maximal response is defined as K0.5 (Like Km, the units of K0.5are
molar-ity; Kmcannot be used to describe these constants, because the protein does not
conform to the Michaelis–Menten model for enzyme kinetics.) The MWC model
accounts for the action of allosteric effectors Positive effectors bind only to the
R state and thus cause a shift of the R st T equilibrium in favor of more R and
thus easier S binding Negative effectors do the opposite; they perturb the R st T
equilibrium in favor of T, the conformation that cannot bind S Note that positive
effectors (allosteric activators) cause a decline in the K0.5for S (signifying easier
binding of S) and negative effectors raise K0.5for S (Figure 15.7) Note that the
MWC model assumes an equilibrium between conformational states, but ligand
binding does not alter the conformation of the protein
Trang 6458 Chapter 15 Enzyme Regulation
The Sequential Model for Allosteric Regulation Is Based
on Ligand-Induced Conformational Changes
An alternative model proposed by Daniel Koshland, George Nemethy, and David
Filmer (the KNF model) relies on the well-accepted idea that ligand binding
trig-gers a change in the conformation of a protein And, if the protein is oligomeric, ligand-induced conformational changes in one subunit may lead to changes in the conformation of its neighbors Such ligand-induced conformational change could cause the subunits of an oligomeric protein to shift from a low-affinity state to a high-affinity state For example, S binding to one monomer may cause the other monomers to adopt conformations with higher affinity for S (Figure 15.8) Inter-estingly, the KNF model also explains how ligand-induced conformational changes could cause subunits of a protein to adopt conformations with little or no affinity
for the ligand, a phenomenon referred to as negative cooperativity The KNF model
is termed the sequential model because subunits undergo sequential changes in
conformation due to ligand binding A comparison of the response of velocity
to substrate concentration for positive versus negative cooperativity is shown in Figure 15.8c
Changes in the Oligomeric State of a Protein Can Also Give Allosteric Behavior
Although the MWC and KNF models are the best-known paradigms for allosteric protein behavior, other models have been put forward For example, instead of R and T, consider a monomer–oligomer equilibrium for an allosteric protein, where only the oligomer binds S and [monomer] [ oligomer] This model strongly
A dimeric protein that can exist in
either of two states: R0 or T0.
This protein can bind three ligands:
1) Substrate (S) : Binds only to
R at site S 2) Activator (A) : A positive effector
that binds only to
R at site F 3) Inhibitor (I) : A negative effector
that binds only to
T at site F
1.0
0.5
0
YS
+A
No A or I
+I
K0.5
Effects of A:
A + R0 R1(A) Increase in number of R-conformers shifts R0 T0
so that T0 R0 (1) More binding sites for S made available.
(2) Decrease in cooperativity of substrate saturation curve.
Effects of I:
I + T0 T1(I) Increase in number of T-conformers (decrease in R0as R0 T0
to restore equilibrium) Thus, I inhibits association of S and A with R by lowering R0 level I increases cooperativity of substrate saturation curve.
[S]
Substrate
R1(S)
Activator
R1(A,S)
Activator
Substrate
R1(A)
T0
T Inhibitor
T1(I)
R0
R
ACTIVE FIGURE 15.7 Allosteric effects: A and I binding to R and T, respectively The linked equilibria lead to changes in the relative amounts of R and T and, therefore, shifts in the substrate saturation curve The parameters of such a system are that (1) S and A (or I) have different affinities for R and T and (2) A
(or I) modifies the apparent K0.5for S by shifting the relative R versus T population Test yourself on the
con-cepts in this figure at www.cengage.com/login.
Trang 7resembles the MWC model In yet another model, we might have a monomeric
pro-tein with distinct binding sites for several different ligands In this case, binding of
ligand A to its site might cause a conformational change such that the protein shows
much greater affinity for S than it would in the absence of A Or, binding of ligand
I might result in a conformational change in the protein such that its affinity for S
is abolished Although the binding of other ligands may affect the affinity of the
monomer for S, S binding cannot show cooperativity in monomeric proteins,
be-cause, unlike oligomers, the monomer has only one binding site for S
It is important to realize that all of these various models are attempts to use
sim-ple concepts to explain the comsim-plex behavior of a protein Although these models
provide reasonable approximations and useful insights, the molecular mechanisms
underlying allostery cannot be expected to conform rigidly to any one of these
mod-els Shortly, we explore the regulated behavior of a real protein (glycogen
phos-phorylase) with these models in mind
the Activity of Enzymes?
Covalent Modification Through Reversible Phosphorylation
As we saw in Figure 15.1, enzyme activity can be regulated through reversible
phos-phorylation; indeed it is the most prominent form of covalent modification in
cel-lular regulation Phosphorylation is accomplished by protein kinases that target
spe-cific enzymes for modification Phosphoprotein phosphatases operate in the
reverse direction to remove the phosphate group through hydrolysis of the
side-chain phosphoester bond Because protein kinases and phosphoprotein
phos-(a) Binding of S induces a conformational change.
Symmetric protein
dimer
S Asymmetric protein dimer
Transmitted conformational change
S
S
If the relative affinities of the various
conformations for S are
(b)
positive cooperativity ensues.
If the relative affinities of the various
conformations for S are
negative cooperativity ensues.
S
(c)
Positive cooperativity No
cooperativity Negative cooperativity
0.2 0.4
0.6
Vmax v
0.8 1.0
3
K0.5
[S]
FIGURE 15.8 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 or lesser 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
chang-ing alignments of nonbonded amino acid residues (c) Theoretical curves for the bindchang-ing of a ligand to a
pro-tein having four identical subunits, each with one binding site for the ligand The fraction of maximal binding is
plotted as a function of [S]/K0.5
Trang 8460 Chapter 15 Enzyme Regulation
phatases work in opposing directions, regulation must be imposed on these con-verter enzymes so that their interconvertible enzyme targets are locked in the de-sired state (active versus inactive) and a wasteful cycle of ATP hydrolysis is avoided Thus, converter enzymes are themselves the targets of allosteric regulation or cova-lent modification
Protein Kinases: Target Recognition and Intrasteric Control
Protein kinases are converter enzymes that catalyze the ATP-dependent phosphoryla-tion of serine, threonine, or tyrosine hydroxyl groups in target proteins (Table 15.2) Phosphorylation introduces a bulky group bearing two negative charges, causing con-formational 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 cat-alytic mechanism based on a conserved catcat-alytic core/kinase domain of approximately
260 amino acid residues (Figure 15.9) Protein kinases are classified as Ser/Thr and/or Tyr specific They also differ in terms of the target proteins that they recognize and phosphorylate; target selection depends on the presence of an amino acid se-quence 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 be-comes 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 15.2) Targeting of protein kinases to particular consensus sequence elements within
pro-teins creates a means to regulate these kinases by intrasteric control Intrasteric con-trol occurs when a regulatory subunit (or protein domain) has a pseudosubstrate
sequencethat mimics the target sequence but lacks an OH-bearing side chain at the right place For example, the cAMP-binding regulatory subunits of PKA (R subunits
in Figure 15.10) possess the pseudosubstrate sequence RRGA*I, and this sequence binds to the active site of PKA catalytic subunits, blocking their activity This
I Ser/Thr protein kinases
A Cyclic nucleotide–dependent
B Ca2-calmodulin (CaM)–dependent Phosphorylase kinase (PhK) OKRKQIS*VRGLO Phosphorylation by PKA Myosin light-chain kinase (MLCK) OKKRPQRATS*NVO Ca2-CaM
D Mitogen-activated protein kinases OPXX(S*/T*)PO Phosphorylation
E G-protein–coupled receptors
-Adrenergic receptor kinase (BARK)
Rhodopsin kinase
II Ser/Thr/Tyr protein kinases
Raf (a protein kinase)
III Tyr protein kinases
A Cytosolic tyrosine kinases (src, fgr, abl, etc.)
B Receptor tyrosine kinases (RTKs) Plasma membrane receptors for hormones
such as epidermal growth factor (EGF) or platelet-derived growth factor (PDGF)
*X denotes any amino acid.
TABLE 15.2 Classification of Protein Kinases
FIGURE 15.9 Protein kinase A is shown complexed with
a pseudosubstrate peptide (orange) This complex also
includes ATP (red) and two Mn 2 ions (yellow) bound at
the active site (pdb id 1ATP).
Trang 9substrate sequence has an alanine residue where serine occurs in the PKA target
se-quence; 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 In other protein kinases, the pseudosubstrate
sequence involved in intrasteric control and the kinase domain are part of the same
polypeptide chain In these cases, binding of an allosteric effector (like cAMP)
in-duces a conformational change in the protein that releases the pseudosubstrate
se-quence from the active site of the kinase domain
The abundance of many protein kinases in cells is an indication of the great
impor-tance of protein phosphorylation in cellular regulation Exactly 113 protein kinase
genes have been recognized in yeast, and 868 putative protein kinase genes have been
identified in the human genome 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
communi-cation (see Chapter 32)
Phosphorylation Is Not the Only Form of Covalent Modification
That Regulates Protein Function
Several hundred different chemical modifications of proteins have been
dis-covered thus far, ranging from carboxylation (addition of a carboxyl group),
acetylation (addition of an acetyl group, see Figure 29.30), prenylation (see
Figure 9.23), and glycosylation (see Figures 7.32–7.39) to covalent attachment
of a polypeptide to the protein (addition of ubiquitin to free amino groups on
proteins; see Figure 31.8), to name just a few A compilation of known protein
modifications can be found in RESID, the European Bioinformatics Institute
online database (http://www.ebi.ac.uk/RESID/) Only a small number of these
co-valent modifications are used to achieve metabolic regulation through reversible
conversion of an enzyme between active and inactive forms Table 15.3 presents a few
examples
+ cAMP
+ 2
C
R2C2
ANIMATED FIGURE 15.10 Cyclic AMP–dependent protein kinase (also known as
PKA) is a 150- to 170-kD R2 C 2 tetramer in mam-malian cells The two R (regulatory) subunits bind
cAMP (KD 3 10 8M); cAMP binding releases
the R subunits from the C (catalytic) subunits.
C subunits are enzymatically active as monomers.
See this figure animated at www.cengage.com/ login.
Reaction Amino Acid Side Chain Reaction (see figure indicated)
Adenylylation Tyrosine Transfer of AMP from ATP to Tyr-OH
(Figure 25.16) Uridylylation Tyrosine Transfer of UMP from UTP to Tyr-OH
(Figure 25.17) ADP-ribosylation Arginine Transfer of ADP-ribose from NADto
Arg (Figure 25.8) Methylation Glutamate Transfer of methyl group from
S-adenosylmethionine to Glu
-carboxyl group
Oxidation-reduction Cysteine (disulfide) Reduction of Cys-S−S-Cys to
Cys-SH HS-Cys (Figure 21.27)
TABLE 15.3 Additional Examples of Regulation by Covalent Modification
Trang 10462 Chapter 15 Enzyme Regulation
Note that three of these types of covalent modification require nucleoside triphos-phates (ATP, UTP) that are related to cellular energy status; another relies on re-ducing potential within the cell, which also reflects cellular energy status
Allosteric Regulation and Covalent Modification?
Glycogen phosphorylase, the enzyme that catalyzes the release of glucose units from glycogen, serves as an excellent example of the many enzymes regulated both by al-losteric controls and by covalent modification
The Glycogen Phosphorylase Reaction Converts Glycogen into Readily Usable Fuel in the Form of Glucose-1-Phosphate
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 15.11) (Because the reaction involves attack by phosphate instead of H2O, it is referred to as a phosphorolysis rather than a
hy-drolysis.) Phosphorolysis produces a phosphorylated sugar product, glucose-1-P,
which is converted to the glycolytic substrate, glucose-6-P, by phosphoglucomutase
(Figure 15.12) In muscle, glucose-6-P proceeds into glycolysis, providing needed energy for muscle contraction In the liver, hydrolysis of glucose-6-P yields glucose, which is exported to other tissues via the circulatory system
Glycogen Phosphorylase Is a Homodimer
Muscle glycogen phosphorylase is a dimer of two identical subunits (842 residues, 97.44 kD) Each subunit contains an active site (at the center of the subunit) and an allosteric effector site near the subunit interface (Figure 15.13) In addition, a
regula-CH2OH O O
CH2OH O O
CH2OH O O
CH2OH O O
+
CH2OH O
CH2OH O O
CH2OH O O
CH2OH O O OPO3–
OH HO
HO
OH
OH OH
OH OH
OH OH
OH OH
OH OH
OH OH
OH OH HO
n
n – 1
-D -Glucose-1-phosphate
P i
residues
FIGURE 15.11 The glycogen phosphorylase reaction.
HOCH2
O
H H
OH OPO3– H
2 –O3POCH2
O
H H
OH OH H
Glucose-1-phosphate Glucose-6-phosphate
FIGURE 15.12 The phosphoglucomutase reaction.