Transcript No Slide Title

COVALENT ENZYME REGULATION
Both reversible and irreversible covalent modification of enzymes play important
roles in regulation of enzyme function. This lecture will cover:
1. Reversible covalent modification. The modulation of enzyme activity by the attachment or
release of small groups plays a very important role in metabolic control. Probably the most
universal, and certainly the most well understood, is the phosphorylation of specific serine,
threonine or tyrosine groups. We will discuss protein phosphorylation, regulation and its
effects on enzyme structure and function
2). Irreversible covalent modification. Proteolytic cleavage of specific peptide bonds is often
used to activate enzymes. Since proteolysis is essentially irreversible, turning the activity off
requires another mechanism, often binding of inhibitory proteins. Examples of enzymes
activated by proteolytic cleavage and the role of enzyme cascades will be discussed.
Reading: Lippincott, ch. 5, section VIII
1) Reversible covalent modifications.
Reversible covalent modifications require expenditure of energy and are
often used in signaling from extracellular messages. In contrast, noncovalent
interactions are reversible with no metabolic energy expended and sense conditions
within a cell. Reversible covalent modifications that are known to alter enzyme
activity include:
a) Phosphorylation of serine, threonine or tyrosine and less frequently
aspartate and histidine residues.
b) Acetylation of lysine or amino terminal groups.
c) Methylation of glutamate or aspartate residues
d) Nucleotidylation of tyrosine residues
e) ADP ribosylation primarily of arginine residues.
Most well understood of these reactions, and probably the most ubiquitous
in eukaryotic cells, is the phosphorylation reaction, which is based on the simple
addition and removal of inorganic phosphate.
Lysine Acetylation
Acetylation of lysines in histones is important in regulation of gene expression.
Addition of the acetyl group to a lysine removes its positive charge, weakening
the binding of histones to the negatively charged DNA which, apparently, results
in a conformation more favorable for transcription
O
NH3+
Ca
CH3
Lysine
S
Acetyl Coenzyme A
H
N
Ca
CoA
C
CoA
CH3
C
O
Acetylated Lysine
HS
Coenzyme A
Protein Phosphorylation: Enzymes catalyzing the transfer of a
phosphate from ATP to a protein are known as kinases and those
catalyzing the hydrolytic removal of the phosphate group are known as
phosphatases.
The energy for
reversible
phosphorylation is
derived from ATP
hydrolysis.
Kinases and Phosphatases come in two major classes
- those that act specifically on serine and threonine
residues and those that act on tyrosine residues.
Ca
HCH
OH OPO32
Serine
Ca
HC
Ca
CH3
HCH
OH OPO32
Threonine
OH
OPO32-
Tyrosine
Regulation of Protein Kinases
Regulation by phosphorylation requires that the kinases and phophatases
must, in turn, be regulated. Regulation of kinases, and probably phosphatases
as well, most often involves one or more of three regulatory strategies:
a) interaction with peptides or subunits whose binding may depend upon
chemical messengers such as calcium or cyclic AMP.
b) phosphorylation itself is a very common mechanism for regulation of
protein kinases (an enzyme that catalyzes this reaction would be known as a
kinase kinase).
c) localization to particular cellular components.
We will provide examples of the first two strategies shortly, using the binding of
cyclic AMP to PKA regulatory subunits as an example of the first strategy and
the effects of phosphorylation on the insulin receptor tyrosine kinase as an
example of the second strategy. An additional important strategy that has
recently become more appreciated is the targeting of kinases to specific cellular
locations. This can be used to limit the effects of kinases with broad specificity
only to desired locations. A number of proteins and protein domains have been
implicated in such targeting.
Phosphorylation in the
regulation of glycogen
metabolism
The role of reversible covalent
modification was first shown to be
important in the control of glycogen
metabolism. Glycogen is used by the
body as a readily mobilized storage of
glucose.
Glycogen
synthase
catalyses synthesis of glycogen while
Glycogen phosphorylase catalyses
the stepwise removal of glucose units
from glycogen.
Synthesis and degradation of glycogen
is coordinated. Shown to the right are
schematic pathways resulting from
epinephrine binding to its receptor on
the plasma membrane. The first three
steps are identical, with both leading to
protein phosphorylation.
However,
phosphorylation
of
phosphorylase
leads to its activation, whereas
phosphorylation of glycogen synthase
leads to its inactivation.
Enzyme Cascades
As is evident from the previous slide, the same protein kinase that
phosphorylates and activates phosphorylase kinase also phosphorylates
glycogen synthase but in this case causes inactivation. Generally
enzymes that are involved in degradation are activated by
phosphorylation and those involved in synthesis are inhibited by
phosphorylation. The activation by an enzyme cascade considerably
amplifies the hormone signal. Three steps are used to activate glycogen
breakdown and two deactivate glycogen synthesis. If glycogen synthase
and phosphorylase were directly controlled by binding epinephrine, more
than one thousand times as much hormone would be needed to elicit the
same response obtained through the cascades.
cAMP dependent kinase (PKA):
The kinase that mediates the hormone signal is
activated by cAMP and is known as cAMP dependent
kinase (cAPK) or protein kinase A (PKA). Although
first discovered in the glycogen metabolism pathway,
PKA is involved in a large number of activities as the
major mediator of cAMP action. PKA specifically
phosphorylates serine or threonine residues preferably
in the following amino acid sequence: Arg - Arg - X Ser (or Thr) - Y, ( where X is a small residue and Y is
a large hydrophobic residue). Most kinases rely
primarily on the amino acid sequence surrounding the
phosphorylation site for their specificity. Activation of
PKA occurs when cAMP binds to the regulatory
subunits. In the absence of cAMP, the regulatory
subunits bind tightly to the catalytic subunits forming a
heterotetramer. A portion of the regulatory subunit
with the sequence: Arg - Arg - Gly - Ala - Ile binds in
the active site of the catalytic subunit. This sequence
matches the preferred sequence except for the actual
phosphorylation site, and thus binds tightly to the
active site without being modified. The binding of
cAMP to the regulatory subunits allosterically causes
dissociation, which allows the catalytic subunits to
attain an active conformation by freeing the active site.
Cyclic AMP
(cAMP)
The crystal structure of the catalytic subunits of PKA
was the first determined for a protein kinase. The
structure has two lobes separated by a deep cleft
that contains the active site (see right). The 240
residue catalytic core shares sequence homology
with hundreds of other kinases suggesting that all
these kinases will have a similar structure. Indeed,
crystal structures for many serine/threonine kinases
and tyrosine kinases demonstrate a common threedimensional structure for the catalytic core of protein
Structure of PKA catalytic
kinases. The structure of PKA was determined
subunit, with bound peptide
bound with a peptide inhibitor revealing structural
inhibitor (dark red)
determinants of sequence specificity. This peptide
has an amphipathic helix that contributes to stability
Asn
Ile
Leu
of the inhibitor-kinase complex and, more
Arg
198
Ala
importantly, contains the sequence Arg-Arg-AsnAla-Ile that binds to the active site. The structure
Arg
shows that both Arg residues are involved in ionic Glu
Leu
Glu
interactions with glutamate residues and the Ile 127
205
Glu
170
residue packs in a hydrophobic groove formed by
230
two Leu residues in the kinase. Thus the specificity Interactions of bound inhibitor
of this kinase for these three residues can be readily (stick bonds) with PKA catalytic
understood.
core residues.
Structural and functional effects of protein phosphorylation
Phosphorylation results in the addition of a doubly negatively charged group (at neutral pH) to a
previously uncharged amino acid. Although this is a small group, it can profoundly impact
protein function. Three different ways this occurs include: a) direct interference with the active
site, b) conformational change in the enzyme or c) creation of binding sites. Examples of
these mechanisms are given below.
a) Direct steric interference is observed in the structures of isocitrate dehydrogenase (IDH)
which show that phosphorylation of Ser 113 in the active site directly interferes with binding of
the isocitrate substrate (see below).
isocitrate
Ser 113
Active site of IDH showing
binding of isocitrate substrate
Phosphate
Ser 113
Active site of IDH showing
phosphorylated Ser 114
b) Enzymatic conformational change upon phosphorylation is dramatically evident from
the structures of the insulin receptor kinase (IRK) determined in the phosphorylated and
unphosphorylated states as shown below. (Figures courtesy of Dr. Stevan Hubbard, NYU.)
Unphosphorylated IRK (left) and phosphorylated IRK (right). The surface of IRK is shown in gray,
except for the N-terminal domain in white in the left figure. The activation loop in unphosphorylated IRK
blocks binding of a substrate polypeptide chain, but moves out of the way, to the right, upon phosphorylation
at three tyrosines. Thus, triple phosphorylation of this loop results in activation of kinase activity.
c) Creation of binding sites – SH2
domains.
Peptides containing a
phosphorylated tyrosine residue are bound
by an important signaling protein module
known as an SH2 (src homology) domain.
These domains are about 100 residues in
length that fold into a central anti-parallel bsheet surrounded by two a-helices. SH2
domains are found in many diverse protein
molecules
and
function
to
bind
phosphorylated tyrosines in a specific
polypeptide sequence. The structure of an
SH2 domain (light gray) with a bound
peptide (green) is shown to the right. The
phosphorylated tyrosine is shown in red,
with the phosphate group in green. Key
residues forming a binding pocket are
shown, including an absolutely conserved
arginine (R32) whose interaction with the
phosphate group is critical for recognizing
phosphorylated tyrosine residues.
R32
In general, a protein kinase is an enzyme that:
A) Catalyzes the transfer of a phosphate group from cAMP to a protein side chain
B) Catalyzes the transfer of a phosphate group from ADP to a protein side chain
C) Catalyzes the transfer of a phosphate group from ATP to a protein side chain
D) Catalyzes the cleavage of a specific peptide bond
E) Catalyzes the removal of a phosphate group from a protein
Which of the following is NOT an important mechanism by which
phosphorylation alters protein function?
A) Phosphorylation can cause direct steric interference by the phosphate at the active site
B) Phosphorylation at serine residues will induce proteolytic cleavage of the peptide bond
amino terminal to the serine
C) Phosphorylation can induce protein conformational changes
D) Phosphorylation at tyrosine residues can create binding sites that are recognized by
SH2 domains
E) Phosphorylation results in the addition of a doubly negatively charged group that can
alter the local protein structure
II) Irreversible covalent modification (limited proteolysis)
In a number of cases, it is necessary to synthesize an enzyme in an inactive state
and activate it later by selective cleavage of one or more peptide bonds. The inactive
precursors are termed zymogens or proenzymes. Proteolytic cleavage generally occurs at
surface loops, rather than secondary structural elements. Generally, the proteolytic site is
amino terminal relative to the active site of the protein. Since the polypeptide is synthesized
in the amino to carboxy direction, the protein can, thus, avoid gaining catalytic activity as it
folds during synthesis.
A) Digestive enzymes
The digestive enzymes are classic examples for which activation of enzyme
activity occurs by selective cleavage. Synthesis as inactive zymogens permits export to the
digestive tract before the destructive catalytic powers of these enzymes are unleashed on
the synthesizing cells.
Gastric and
pancreatic
digestive
enzymes
Site of synthesis
Zymogen
Active Enzyme
Stomach
Pepsinogen
Pepsin
Pancreas
Chymotrypsinogen
Chymotrypsin*
Pancreas
Trypsinogen
Trypsin*
Pancreas
Procarboxypeptidase
Carboxypeptidase
Pancreas
Proelastase
Elastase*
*Serine Protease
Chymotrypsin, trypsin and elastase are examples of the major class of proteases
known as serine proteases (discussed in lecture 15) which are so named because they have
a highly reactive serine at the active site that is essential for catalytic activity. Different
members of the serine protease family have very similar catalytic sites, but distinct specificity
due to flanking regions that bind various amino acid side chains with differing affinity.
The first step in the activation of
pancreatic zymogens is a very specific
cleavage of a small amount trypsinogen by
enteropeptidase.
This occurs after
trypsinogen secretion from the pancreas into
the duodenum. These newly formed trypsin
molecules
activate
other
trypsinogen
molecules and other zymogens.
Activation of enzymatic activity does not require major changes in the protein structure.
Chymotrypsinogen is activated by cleavage of the peptide bond between residues 15 and
16, with the N-terminal peptide remaining attached to the rest of the protein due to a
disulfide linkage. (Additional cleavages can take place but are not essential for catalytic
activity.) Cleavage does not lead to large molecular changes, but rather to subtle changes
that alter the active site geometry so as to maximally stabilize the transition state.
Inactivation of digestive enzymes
Since activation by selective peptide bond cleavage is irreversible, alternate
mechanisms must exist for turning off enzyme activity. Specific protease inhibitors are
available for this function. Pancreatic trypsin inhibitor (PTI) is a small (6-kDa) protein
that binds to trypsin tightly in its active site. Binding is very tight due to precise
structural complementarity between PTI and the active site of trypsin.
A much larger protein, the 53 kDa a1-antitrypsin, plays a
similar role in the inactivation of elastase. Elastase is secreted by
neutrophils as part of the inflammatory process. In order to restrict
its activity to the site of infection, it is turned off by a1-antitrypsin.
The importance of its role is apparent from misfolding mutants.
One mutation (glu 53 -> lys) results in reduced secretion from the
liver, lowering the serum level of a1-antitrypsin to 15% of that in
normal individuals.
(The lowered secretion stems from the
pathological polymerization of the mutant, causing cirrhosis of the
liver, as discussed in lecture 5.) The resulting excess elastase
activity in the serum destroys the alveolar walls in the lungs and
leads to emphysema. Cigarette smoking increases the propensity
of heterozygotes for the above mutation to develop emphysema,
as a result of oxidation of methionine 358 of a1-antitrypsin (see
right). The addition of a single oxygen atom in this residue
drastically decreases the affinity of the inhibitor for elastase.
-CH2- CH2 – S – CH3
Methionine
Oxidation
-CH2- CH2 – S – CH3
O
Methionine sulfoxide
B) Blood Clotting. Blood clotting results from the participation of nearly 20 different
substances involved in two different cascades of proteolytic reactions. The use of cascades
serve to amplify small stimuli to major physiological responses. The two cascades are
known as the intrinsic and extrinsic pathways. The intrinsic pathway covers five proteolytic
reactions and is mediated by components found in the plasma. The extrinsic pathway
comprises four proteolytic steps and includes factors found in tissues. Both pathways meet
at the conversion of prothrombin to thrombin which then converts fibrinogen to fibrin.
The active clotting
factors are, except for
fibrin and factor XIIIa,
serine proteases
similar to the members
of the trypsin family.
The final purpose of
these cascades is the
conversion of fibrinogen
to fibrin to form a clot.
Formation of Fibrin Clot:
The ultimate purpose of the blood clotting pathways is to trigger the
conversion of fibrinogen into fibrin which then polymerizes to form a clot. Fibrinogen is very abundant,
comprising 2-3% of plasma protein, and is formed from three pairs of polypeptide chains into a highly
disulfide linked elongated complex (~450A in length).
Crystal structure of bovine
fibrinogen. The aA chains
are shown in green, bB chains
in blue and g in red.
Schematic diagram of fibrinogen. At the molecular center are four “fibrinopeptides” (two A and two
B) at the amino end of the a and b chains whose abundance of negative charge inhibits polymerization.
Conversion to fibrin results from thrombin cleavage at specific Arg-Gly peptide bonds, which releases these
peptides and results in the center region becoming more positively charged.
-6
-6
Thrombin cleavage
-4
+5
-4
Fibrin Clot. Following removal of the negatively charged A and B fibrinopeptides, charge interactions
between fibrin molecules stabilize the formation of the arrangement illustrated below. This half-staggered
arrangement, with a fiber repeat of 225Å, is the fundamental assemblage of the fibrin clot.
The fibrin clots are considered "soft clots" and can be turned into more stable "hard clots"
cross-linking lysine and glutamine residues in a transamidation reaction by factor XIIIa.
(Factor XIIIa is also known as fibrin-stabilizing factor (FSF) or transglutaminase.)
O
Lys-CH2- CH2 -CH2- CH2 -NH2
+
Activated
FSF
NH2 -C -CH2- CH2 - Gln
NH3
O
Lys-CH2- CH2 -CH2- CH2 -NH -C -CH2- CH2 - Gln
Thrombin catalyzes the cleavage of the Arg-Gly bond in fibrinogen that initiates clot
formation. Its structure shows very striking similarity with those of the pancreatic serine
proteases especially in the active site. The major difference is its extreme specificity in
cleaving only certain Arg - Gly bonds.
Because of the amplification effects of the cascade reactions, the early factors are only
present at very small concentrations (mg/ml) compared with the 3mg/ml for fibrinogen. This,
along with their lack of stability, has complicated analysis of these proteins. Much of the early
knowledge of the clotting reaction came from bleeding disorders. The most well known of
these is classic hemophilia, a sex-linked disorder in which Factor VIII is missing or has a very
reduced activity. This factor stimulates the activation of Factor X by Factor IX. Treatment of
hemophiliacs used to require that large amounts of blood be fractionated to obtain reasonable
quantities of coagulation factors. This was expensive and risky. The availability of
recombinant clotting factors now minimizes these risks.
Blood clots are only meant to be temporary patches. Once structural integrity is returned to
the damaged area, they must be dissolved. The protein responsible for lysis of the clot is, once
again, a serine protease, this time one named plasmin. The open mesh-like structure of a blood
clot allows ready access to plasmin, which is formed by the proteolytic cleavage of plasminogen.
Several serine proteases are involved in the
activation of plasminogen including urokinase
and tissue-type plasminogen activator (TPA).
Plasminogen activators are of considerable
medical significance and are used to
promote rapid dissolving of blood clots
responsible for heart attacks and strokes.
Both recombinant TPA and streptokinase
have been successfully used for this
purpose.
The pancreatic digestive enzymes are activated by:
A) Cleavage of specific peptide bonds by trypsin
B) Binding of BPG to basic residues in the subunit interface
C) Cleavage of C-terminal residues by carboxypeptidase
D) Phosphorylation of serine and threonine residues
E) Methylation of aspartate residues
TPA is of medical importance because it:
A) Directly dissolves fibrin based blood clots.
B) Cleaves plasminogen to form plasmin which then dissolves blood clots.
C) Phosphorylates and activates plasminogen which then dissolves blood clots.
D) Prevents massive bleeding by stimulating blood clotting.
E) Assists plasmin in dissolving blood clots by opening up cleavage sites.
Summary Points on Covalent Enzyme Regulation:
1) Know the reactions catalyzed by protein kinases and phosphatases.
2) Understand the amplification effect of enzyme cascades.
3) Know the basis for activation of cAMP dependent Kinase (PKA).
4) Know that there are three important basic mechanisms by which
phosphorylation alters protein function including: steric interference,
conformational change as a result of introduction of a doubly negatively charged
group and creation of binding sites.
5) Understand that activation of digestive enzymes and blood clotting factors by
proteolysis may be mediated by localized subtle structural changes that have
large functional consequences.
6) Know that enzymes activated by proteolysis are usually inactivated by binding
inhibitors.
7) Know what is meant by a serine protease.
8) Know that activation of fibrinogen to form fibrin clots occurs as a result of
proteolysis of specific Arg-Gly bonds by thrombin.
9) Know that TPA is a serine protease that promotes lysis of blood clots by
proteolytic cleavage of plasminogen to form plasmin