• Proteinases (Proteases)

• Cleave proteins
• Four Families-based on functional
• group in the active site
• Serine
Cysteine
• Aspartic
• Metallo
 Serine Proteinases
• Chymotrypsin--mammalian digestive
enzyme which
cleaves tryptophan, tyrosine,
phenylalanine, and
methionine
• How do proteinases work?
How do serine proteinases cleave peptide bonds
• proteinases allow this reaction
to occur?
• Four important
• structural features of
• serine proteinase
• – Catalytic triad
• – Oxyanion hole
• – Nonspecific bonding
• – Specificity pocket
 CHYMOTRYPSIN

• Two domains
• – ~120 a. a.
each
• – Antiparallel
betabarrel
 Carboxypeptidase

• A peptide substrate binds at the active site of the enzyme. X-ray structures of the
enzyme with and without a competitive inhibitor show a large conformational change
at the active site when inhibitor or substrate is bound. Without inhibitor, several
waters occupy the active site. When inhibitor and presumably substrate are bound,
the water leaves (which is entropically favored), and Tyr 248 swings around from near
the surface of the protein in the absence of a molecule in the active site to interact
with the carboxyl group of the bound molecule, a distance of motion equal to about
1/4 the diameter of the protein. This effectively closes off the active site and expels
the water. A Zn2+ ion is present at the active site. It is bound by His 69, His 196, Glu
72, and finally a water molecule as the fourth ligand. A hydrophobic pocket which
interacts with the phenolic group of the substrate accounts for the specificity of the
protein.
• In the catalytic mechanism, Zn2+ helps polarize the labile amide bond, while Glu 270,
acting as a general base, which along with Zn2+ helps promote dissociation of a
proton from the bound water, making it a better nucleophile. Water attacks the
elctrophilic carbon of the sessile bond, with Glu 270 acting as a general base catalyst.
The tetrahedral intermediate then collapses, expelling the leaving amine group, which
picks up a proton from Glu 270, which now acts as a general acid catalyst. People
used to believe that Tyr 248 acted as a general acid, but mutagenesis showed that
Tyr 248 can be replaced with Phe 248 without significant effect on the rate of the
reaction.
 CarboxypeptidaseA

• • Carboxypeptidase A (CPA) hydrolyzes C-terminal amino

• acids of polypeptide chains, exhibiting a preference
• towards substrates with large hydrophobic side chains
• (such as Phe)
• – metalloexopeptidase with 1 mole Zn2+ per mole enzyme
• – 307 amino acids, 34.5 kDa
• – excellent crystal structure of free form (1.54 Å resolution)
• • also structures of substrate analogs, TS analogs, slowly
hydrolyzed
• substrates, and product analogs complexed with CPA
• – Zn2+ coordinated by Glu-72, His-69, His-196, and H2O
 Mechanism for CPA

• Possible mechanisms for CPA

• • 2 possible mechanisms were proposed for the role of Glu-
• 270
• – Activated water mechanism: (Glu-270 as a general base) Glu-
270,
• perhaps in conjunction with Zn2+, activates water as a nucleophile
to
• hydrolyze the peptide bond
• – Nucleophilic mechanism: (Glu-270 as a nucleophile) Glu-270
acts
• as a nucleophile directly to hydrolyze peptide bonds, forming an
• anhydride intermediate
• • The crystal structure of CPA does not distinguish between
• these two mechanisms (controversial whether Glu-270 is
• close enough for nucleophilic attack)
• – 18O isotope labeling experiments helped to determine peptide
• hydrolysis mechanism (reverse reactions with 18O labeled acid)
 CPA Mechanism

• The carbonyl that is H-bonded to Arg-

127 is polarized
• Zn2+-bound H2O can act as a
nucleophile, Glu-270 acts as general
base
• tetrahedral intermediate is stabilized
by Zn2+– aldehyde and ketone
analogs are found in their
• hydrated forms in CPA crystal
structures
• • Tyr-248 or Glu-270 act as a general
acid to
• protonate leaving group amine
 Lysozyme

• This enzyme, found in cells and secretions of vertebrates but also in viruses
which infect bacteria, cleaves peptidoglycan GlcNAc (b 1->4) MurNAc
repeat linkages (NAG-NAM) in the cell walls of bacteria and the GlcNAc (b
1->4) GlcNAc (poly-NAG) in chitin, found in the cells walls of certain fungi.
Since these polymers are hydrophilic, the active site of the enzyme would
be expected to contain a solvent-accessible channel into which the polymer
could bind. The crystal structures of lysozyme and complexes of lysozyme
and NAG have been solved to high resolution. The inhibitors and
substrates form strong H bonds and some hydrophobic interactions with the
enzyme cleft. Kinetic studies using (NAG)n polymers show a sharp
increase in kcat as n increases from 4 to 5. The kcat for NAG6 and (NAG-
NAM)3 are similar. Models studies have shown that for catalysis to occur,
(NAG-NAM)3 binds to the active site with each sugar in the chair
conformation except the fourth which is distorted to a half chair form, which
labilizes the glycosidic link between the 4th and 5th sugars. Additional
studies show that if the sugars that fit into the binding site are labeled A-F,
then because of the bulky lactyl substituent on the NAM, residues C and E
can not be NAM, which suggests that B, D and F must be NAM residues.
Cleavage occurs between residues D and E.
 Lysozyme
• Catalysis by the enzyme involves Glu 35 and Asp 52 which are in
the active site. Asp 52 is surrounded by polar groups but Glu 35 is
in a hydrophobic environment. This should increase the apparent
pKa of Glu 35, making it less likely to donate a proton and acquire a
negative charge at low pH values, making it a better general acid at
higher pH values. The general mechanism appears to involve:
• binding of a hexasaccharide unit of the peptidoglycan with
concomitant distortion of the D NAM.
• protonation of the sessile acetal O by the general acid Glu 35 (with
the elevated pKa), which facilitates cleavage of the glycosidic link
and formation of the resonant stabilized oxonium ion.
• Asp 52 stabilizes the positive oxonium through electrostatic
catalysis. The distorted half-chair form of the D NAM stabilizes the
oxonium which requires co-planarity of the substituents attached to
the sp2 hybridized carbon of the carbocation resonant form (much
like we saw with the planar peptide bond).
• water attacks the stablized carbocation, forming the hemiacetal with
release of the extra proton from water to the deprotonated Glu 35
reforming the general acid catalysis.
 Lysozyme

• Binding and distortion of the D substituent of the

substrate (to the half chair form as shown above) occurs
before catalysis. Since this distortion helps stabilize the
oxonium ion intermediate, it presumably stabilizes the
transition state as well. Hence this enzyme appears to
bind the transition state more tightly than the free,
undistorted substrate, which is yet another method of
catalysis.
• pH studies show that side chains with pKa's of 3.5 and
6.3 are required for activity. These presumably
correspond to Asp 52 and Glu 35, respectively. If the
carboxy groups of lysozyme are chemically modified in
the presence of a competitive inhibitor of the enzyme,
the only protected carboxy groups are Asp 52 and Glu
35.