Clinical%20Aspects%20of%20Biochemistry

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• Clinical Aspects of Biochemistry
• Proteins and Disease
• Serine proteases
• Serine proteases - summary
• Zymogen activation and its control
• Leukocyte elastase
• Prohormone convertases

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• 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

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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
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ZYMOGEN ACTIVATION
From Stryer
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CHYMOTRYPSINOGEN ACTIVATION
From Stryer
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MECHANISM OF ACTIVATION OF CHYMOTRYPSINOGEN Cleav
age 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
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From Stryer
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ACUTE PANCREATITIS (PREMATURE ACTIVATION OF
ZYMOGENS)

• Triggered by trauma to pancreas
• Normally prevented by
• Producing enzymes as zymogens
• Storing zymogens in protease-resistant vesicles
• Presence of pancreatic trypsin inhibitor (PTI)

PTI Protein of Mr 6K. Binds tightly to active
site, like substrate, but not cleaved
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From Stryer
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• 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.
• a1-proteinase inhibitor and other serpins may
  also be involved in other degenerative diseases.
  Much effort to develop specific inhibitors of the
  enzyme

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Anti-protease action of Serpins
From Carrell Corral (2003)
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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.
-Gly-Asp-Ser-Gly-. esterases
-Gly-Glu-Ser-Ala- Evolutionary tree describing
these relationships
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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
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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

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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

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SUBTILISIN AND FURIN
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PC1 DEFICIENCY IN MAN
Mutations in PC1 can lead to severe PC1
deficiency. Symptoms
Elevated proinsulin and ACTH precursors Obesity Hy
pogonadotropic hypogonadism