Transcript 15.1 – What Factors Influence Enzymatic Activity?

Chapter 15
Enzyme Regulation
Biochemistry
by
Reginald Garrett and Charles Grisham
Essential Question
1. What are the properties of regulatory
enzymes?
2. How do regulatory enzymes sense the
momentary needs of cells?
3. What molecular mechanisms are used to
regulate enzyme activity?
Outline of Chapter 15
1. What Factors Influence Enzymatic Activity?
2. What Are the General Features of Allosteric
Regulation?
3. Can Allosteric Regulation Be Explained by
Conformational Changes in Proteins?
4. What Kinds of Covalent Modification
Regulate the Activity of Enzymes?
5. Is the Activity of Some Enzymes Controlled
by Both Allosteric Regulation and Covalent
Modification?
15.1 – What Factors Influence
Enzymatic Activity?
• The activity displayed by enzymes is affected
by a variety of factors, some of which are
essential to the harmony of metabolism
• Two of the more obvious ways to regulate the
amount of activity are
1. To increase or decrease the number of enzyme
molecule (enzyme level)
2. To increase or decrease the activity of each
enzyme molecule (enzyme activity)
A general overview of factors influencing
enzyme activity includes the following
considerations
1. Rate depends on substrate availability
2. Rate slows as product accumulates
3. Genetic controls (transcription regulation) induction and repression (enzyme level)
4. Enzyme activity can be regulated allosterically
5. Enzyme activity can be regulated through
covalent modification
6. Zymogens, isozymes and modulator proteins
may play a role
Figure 15.1 Enzyme regulation by reversible covalent
modification.
Zymogens
Figure 15.2
Proinsulin is an 86residue 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.
Serine protease:
Kallikrein
VIIa
IXa
Xa
XIa
XIIa
Thronbin
Figure 15.4 The cascade of activation steps leading to blood clotting.
Rich in negative
charge
formation of a blood clot.
Isozymes
1. LDH M is a A4 homotetramer in muscle
2. LDH H is a B4 homotetramer In heart
3. Different tissues express different isozyme forms, as appropriate to particular metabolic
needs
Figure 18.21 (b) In oxygendepleted muscle, NAD+ is
regenerated in the lactate
dehydrogenase reaction.
1. LDH M works best in pyruvate-to-lactate direction.
2. LDH H works best in lactate-to-pyruvate direction
• Modulator proteins are another way that
cells mediate metabolic activity
– cAMP-dependent protein kinase
– Phosphoprotein phosphatase inhibitor-I
15.2 – What Are the General
Features of Allosteric Regulation?
Action at "another site"
• Allosteric regulation acts to modulate enzymes
situated at key steps in metabolic pathways
Enz 1
Enz 2
Enz 3
Enz 4
Enz 5
A  B  C  D  E  F
• F, the essential end product, inhibits enzyme 1,
the first step in the pathway
• This phenomenon is called feedback inhibition
or feedback regulation
•
Regulatory enzymes have certain exceptional
properties
1. Their kinetics do not obey the Michaelis-Menten
equation
– Their v versus [S] plots yield sigmoid- or Sshaped curve
– A second-order (or higher) relationship between v
and [S]
– Substrate binding is cooperative
• Regulatory enzymes have certain exceptional
properties
1. Their kinetics do not obey the Michaelis-Menten
equation
2. Inhibition of a regulatory enzyme by a feedback
inhibitor does not conform to any normal
inhibition pattern
- Feedback inhibitor (Product) bears the structural
similarity to A, may bind to substrate-binding site
- If product acts at a site other than the substrate-binding
site—called Allosteric inhibition
• Regulatory enzymes have certain exceptional
properties
1. Their kinetics do not obey the Michaelis-Menten
equation
2. Inhibition of a regulatory enzyme by a feedback
inhibitor does not conform to any normal
inhibition pattern- Allosteric inhibition
3. Some effector molecules exert negative effects on
enzyme activity, other effectors show stimulatory,
or positive, influences on activity
4. Oligomeric organization
5. The regulatory effects exerted on the enzyme’s
activity are achieved by comformational changes
occurring in the protein when effector metabolites
bind
15.3 – Can Allosteric Regulation Be
Explained by Conformational Changes in
Proteins?
Symmetry model : Two conformational states
• Monod, Wyman, Changeux (MWC) Model:
allosteric proteins can exist in two states: R
(relaxed) and T (taut)
• In this model, all the subunits of an oligomer must
be in the same state (R or T)
• T state predominates in the absence of substrate S
R1  R0  T0  T1
L= T0 / R0
L is equilibrium constant
• Although the relative [R0] concentration is small,
S will bind ‘only’ to R0, forming R1
– S binds much tighter to R than to T
• S-binding drives the conformation transition,
T0  R0 (because R0 becomes R1 , R0 decreases)
• Cooperativity is achieved because S binding
increases the population of R, which increases the
sites available to S
• K0.5 (Km)
• Ligands such as S are positive homotropic
effectors
• Molecules that influence the binding of
something other than themselves are
heterotropic effectors
– Positive heterotropic effectors or allosteric
avtivators (T0  R0)
– negative heterotropic effectors or allosteric
inhibitors (R0  T0)
The sequential model
• Proposed by Koshland, Nemethy, and Filmer (the KNF
model) relies on the idea that ligand binding triggers a
conformation change in a protein
• If the protein is oligomeric, ligand-induced conformation
changes in one subunit may lead to conformation
changes in adjacent subunits
The notable difference between the MWC and
KNF model
1. In the MWC model, the different conformations have
different affinities for the various ligand
2. the KNF model is based on ligand-induced
conformation changes
Figure 15.8 The KoshlandNemethy-Filmer sequential
model for allosteric
behavior.
15.4 What Kinds of Covalent Modification
Regulate the Activity of Enzymes?
Covalent modification through reversible
phosphorylation
• This is the most prominent form of covalent
modification in cellular regulation
1. Phosphorylation is accomplished by protein
kinases—each protein kinase targets specific
proteins for phosphorylation
2. Phosphoprotein phosphatases catalyze the reverse
reaction – removing phosphoryl groups from
proteins
• Protein kinases and phosphatases work in opposing
directions
Figure 15.1 Enzyme regulation by reversible covalent
modification.
• Protein kinases phosphorylate Ser, Thr, and Tyr
residues in target proteins (Table 15.2)
• Phosphorylation introduces a bulky group
bearing two negative charges, causing
conformational changes that alter the target
protein’s function
• Protein kinases are classified as Ser/Thr and/or
Tyr specific
• Kinases are often regulated by intrasteric control
Phosphorylation is Not the Only Form of
Covalent Modification that Regulates Protein
Function
• Several hundred different chemical modifications of
proteins have been discovered
• Only a few of these are used to achieve metabolic
regulation through reversible conversion of an enzyme
between active and inactive forms
• A few are summarized in Table 15.3
• Three of the modifications in Table 15.3 require
nucleoside triphosphates (ATP, UTP) that are related to
cellular energy status
15.5 Is the Activity of Some Enzymes
Controlled by Both Allosteric Regulation
and Covalent Modification?
Glycogen phosphorylase cleaves glucose units
from nonreducing ends of glycogen
• A phosphorolysis reaction (Fig 15.12)
• Regulated both by allosteric control and by
covalent modification
• Glucose-1-P is converted to glucose-6-P
• In muscle, proceeds into glycolysis (chapter 18)
• In liver, hydrolysis of glucose-6-P yields
glucose
Figure 15.12 The glycogen phosphorylase reaction.
Figure 15.13 The phosphoglucomutase reaction.
• Muscle glycogen phosphorylase is a dimer of
two identical subunits (842 residues)
– Each subunit contains a pyridoxal phosphate (PLP)
cofactor covalently linked (Lys-680)
– A tower helix (residues 262 to 278)
– An allosteric effector site near the subunit interface
– A regulatory phosphorylation site (Ser14)
– A glycogen binding site
– An active site
Glycogen Phosphorylase Activity is
Regulated Allosterically
• Cooperativity in substrate binding (15.15a)
– Inorganic phosphate (Pi) is a positive homotropic
effector
• ATP and Glucose-6-P are feedback inhibitors,
and negative heterotropic effectors (i.e.,
allosteric inhibitor) (15.15b)
• AMP is a positive heterotrophic effector (i.e.,
allosteric activator) (15.15c)
• ATP and AMP bind to the same site (reciprocal
regulation in the cellular concentration of ATP
and AMP
Figure 15.14 v versus S curves for glycogen phosphorylase.
The response to the concentration of the substrate phosphate (Pi).
ATP is a feedback inhibitor.
AMP is a positive effector. It binds at the same site as ATP.
MWC model
• The active form of the enzyme is designated the R
state
• The inactive form of the enzyme is denoted as the T
state
• AMP promotes the conversion to the active state
• ATP, glucose-6-P, and caffeine favor conversion to
the inactive T state
• A significant change occurs at the subunit interface
between the T (Asp283) and R state (Arg569) –Pi
• This conformational change at the subunit interface is
linked to a structural change at the active site that
affects catalysis (fig 15.17)
Figure 15.16
The mechanism of
covalent modification
and allosteric regulation
of glycogen
phosphorylase. The T
states are blue and the
R states blue-green.
Regulation of GP by Covalent
Modification
• In 1956, Edwin Krebs and Edmond Fischer
showed that a ‘converting enzyme’ could
convert phosphorylase b to phosphorylase a
• Three years later, Krebs and Fischer show
that this conversion involves covalent
phosphorylation (Figure 15.16)
• Phosphorylation of Ser14 causes a dramatic
conformation change in phosphorylase
(Figure 15.17)
Figure 15.17 The major
conformational change that
occurs in the N-terminal
residues upon
phosphorylation of Ser14.
Ser14 is shown in red.
N-terminal conformation of
phosphorylated enzyme
(phosphorylase a): yellow.
N-terminal conformation of
unphosphorylated enzyme
(phosphorylase b): cyan.
This phosphorylation is mediated
by an enzyme cascade
• Cyclic AMP (cAMP) is the intracellular
agent of extracellular hormones - thus a
‘second messenger’
• Hormone binding stimulates a GTP-binding
protein (G protein), releasing G(GTP)
• Binding of G(GTP) stimulates adenylyl
cyclase to make cAMP
Figure 15.18 The hormone-activated
enzymatic cascade that leads to activation
of glycogen phosphorylase.
Figure 15.19 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 15.20 Hormone binding to its
receptor leads via G-protein
activation to cAMP synthesis.
Adenylyl cyclase and the hormone
receptor are integral membrane
proteins; Gα and Gβγ are
membrane-anchored proteins.
Hemoglobin
•
•
•
•
•
A classic example of allostery
Hemoglobin (Hb) and myoglobin (Mb) are
oxygen transport and storage proteins
Compare the oxygen binding curves for
hemoglobin and myoglobin
Myoglobin is monomeric; hemoglobin is
tetrameric
Mb: 153 residues
Hb: two s of 141 residues, 2 bs of 146 (2b2)
Hemoglobin Function
Hb must bind oxygen in lungs and
release it in capillaries
• Adjacent subunits' affinity for oxygen
increases
• This is called positive cooperativity
Figure 15.21 O2-binding curves for hemoglobin and myoglobin.
The Bohr Effect
•
•
•
•
Competition between oxygen and H+
Discovered by Christian Bohr
Binding of protons diminishes oxygen
binding
Binding of oxygen diminishes proton
binding
Important physiological significance
Figure 15.33 The oxygen saturation curves for myoglobin and for hemoglobin at five
different pH values: 7.6, 7.4, 7.2, 7.0, and 6.8.
Bohr Effect II
Carbon dioxide diminishes oxygen binding
• Hydration of CO2 in tissues and
extremities leads to proton production
• These protons are taken up by Hb as
oxygen dissociates
• The reverse occurs in the lungs
The ionic binding of BPG to the two βsubunits of Hb. BPG lies at the center of
the cavity between the two β-subunits.
• Fetal Hb differs from adult Hb – with γ-chains in place of βchains – and thus a α2γ2 structure
• Fetal Hemoglobin Has a Higher Affinity for O2 Because it
has a Lower Affinity for BPG