Download serine proteases

Clinical Aspects of Biochemistry
Proteins and Disease
Serine proteases
1. Serine proteases - summary
2. Zymogen activation and its control
3. Leukocyte elastase
4. Prohormone convertases
SERINE PROTEASES - A SUMMARY
You should know most of this from previous lectures (!)
1. Serine proteases are members of a large group of proteolytic enzymes, all of
which have at their active site a serine residue which plays a crucial part in the
enzymic activity. All cleave peptide bonds, by a similar mechanism of action. They
differ in their specificity and regulation.
2. Serine proteases include:
•the pancreatic proteases, trypsin, chymotrypsin and elastase,
•various tissue/intracellular proteases such as leukocyte elastase
•prohormone convertases (PCs),
•enzymes of the blood clotting cascade (see next lecture)
•some enzymes of 'complement’ (immunology lectures?)
3. Many serine proteases are synthesized as inactive precursors (zymogens) which
are activated by proteolysis
4. The mechanism of action of serine proteases involves:
A catalytic triad (Asp...His...Ser) which converts the active site serine to a
powerful nucleophile.
An oxyanion pocket which facilitates the formation of the tetrahedral
transition state
Acylation of the active site serine to give a covalent intermediate
5. Specificity arises from the presence of a pocket near the active site which
binds amino acid side chains of the substrate; the structure of this pocket varies
from one enzyme to the next.
Three main topics will be covered in more detail in this lecture:
a) Zymogen activation and its control. Premature activation can cause acute
pancreatitis
b) Leukocyte elastase and its role in pulmonary emphysema
c) Prohormone convertases - relation to other proteases, biological role,
possible association with disease
ZYMOGEN ACTIVATION
From Stryer
CHYMOTRYPSINOGEN ACTIVATION
From Stryer
MECHANISM OF ACTIVATION OF CHYMOTRYPSINOGEN
Cleavage of Arg15 -Ile16 leads to conformational changes:
1. Gives new COO- and +NH3 groups.
2. New +NH3 turns in and interacts with Asp194 in interior of molecule. This amino
group must be protonated for enzyme to be active.
3. Interaction between these +ve and -ve charges in a non-polar region triggers
conformational changes:
Met192 moves from buried (in zymogen) to surface (in enzyme).
Residues 187 and 193 become more extended.
These conformational changes give rise to the substrate binding site (the cavity
doesn't exist in the zymogen)
4. Transitional complex is stabilised by H-bonds that can only form in the active
enzyme (main chain NH of Gly193). I.e. the oxyanion pocket becomes functional
5. Other conformational changes are minor
From Stryer
ACUTE PANCREATITIS
(PREMATURE ACTIVATION OF ZYMOGENS)
Triggered by trauma to pancreas
Normally prevented by:
1. Producing enzymes as zymogens
2. Storing zymogens in protease-resistant vesicles
3. Presence of pancreatic trypsin inhibitor (PTI)
PTI
Protein of Mr 6K.
Binds tightly to active site, like substrate, but not cleaved
From Stryer
LEUKOCYTE ELASTASE & 1-PROTEINASE INHIBITOR
• Leukocyte elastase is involved in the inflammatory process
• Controlled by 1-proteinase inhibitor, a serum protein secreted by
liver. A member of the serpin (serine protease inhibitor) protease
family
• Variants of inhibitor with reduced activity are associated with
pulmonary emphysema (degenerative lung disease - damage to
elastin)
• Smokers also have decreased activity of the inhibitor (and lung
damage) because of the oxidation of active site Met to Met
sulphoxide. This is especially a problem in heterozygotes for the
defective inhibitor gene.
• 1-proteinase inhibitor and other serpins may also be involved in
other degenerative diseases. Much effort to develop specific
inhibitors of the enzyme
Anti-protease action of Serpins
From: Carrell & Corral (2003)
CH3
|
S
|
CH2
|
CH2
|
...NH.CH.CO…
CH3
|
S==O
|
CH2
|
CH2
|
...NH.CH.CO…
Methionine
Methionine sulphoxide
SERINE PROTEASE FAMILY
Serine proteases like trypsin, chymotrypsin and elastase
are structurally related, and also related to esterases (such as
butyryl esterase, liver aliesterase, acetyl cholineesterase)
Active site sequence::
trypsin etc.
esterases
-Gly-Asp-Ser-Gly-.
-Gly-Glu-Ser-Ala-
Evolutionary tree describing these relationships:
cholinesterase etc
trypsin
elastase
A
) chymotrypsin
B
thrombin
ANOTHER SERINE PROTEASE FAMILY
In bacteria and fungi, some serine proteases are trypsin-like, but
others are not, e.g. subtilisin and aspergillus protease. These
have active site sequence:
Thr-Ser-Met (c.f.Asp-Ser-Gly for trypsin)
In sequence and tertiary structure subtilisin and aspergillus
protease are similar, but have no similarity to trypsin etc. But
charge relay is similar:
Asp32....His64....Ser221 (c.f.Asp102....His59....Ser195 in chymotrypsin)
So convergent evolution
Until recently no mammalian homologue of subtilisin was known,
but recently prohormone/proprotein convertases have proved to
be subtilisin-like
HUMAN PROPROTEIN CONVERTASES (PCs)
• Family of at least 7 similar subtilisin like serine proteases
• Convert proinsulin to insulin and process many peptide
hormone, neuropeptide and growth factor precursors
• Specificity varies (allowing differential processing), but
most cleave at paired basic residues, which sometimes have
to be presented on a b-turn
• Expressed differentially in various tissues, especially
endocrine glands and neural tissue
• Produced as precursors, which themselves have to be
processed (autocatalytically?)
• Also involved in processing various virus and toxin protein
precursors
PRECURSORS OF PC1 AND PC2
• C-terminal domain (P domain) can function in sorting and
membrane attachment
• Pro region can also act as inhibitor of enzyme
SUBTILISIN AND FURIN
Furin
Subtilisin
Ser
Ser
His
His
Asp
Asp
PC1 DEFICIENCY IN MAN
Mutations in PC1 can lead to severe PC1 deficiency. Symptoms:
Elevated proinsulin and ACTH precursors
Obesity
Hypogonadotropic hypogonadism