Biochemistry 6/e

1
Catalytic strategies
What are the sources of catalytic power and
selectivity of enzymes? We investigate here a
specific class of enzymes Ser-proteases. Mechani
sm of action of these enzymes got determined by a
great selection of state-of-the-art biochemical
and biophysical approaches including structure
determination or site-directed mutagenesis. In
each class of Es, comparison between class
members reveal how Es and their ASs have evolved
and been refined to deliver specific tasks.
Struc- tural and mechanistic comparisons of E
actions provide us with insight to evolution of
Es. These insights lead to design more potent
drugs.
2
Types of catalytic strategies Covalent
catalysis AS contains a reactive group, often a
nucleophile, that temporarily covalently binds to
S. Acid-base catalysis a H donor or acceptor
plays role, e.g. in chymotrypsin a His is a base
catalyst to enhance the nucleophilicity of
Ser. Catalysis by approximation based simply on
binding the two Ss in a single binding
surface. Metal ion catalysis Metal ions can act
several ways, e.g. make nucleophiles like OH- by
direct coordination (like Zn2 in carbonic
anhydrase), serve as electrophile stabilizing a
charge in an intermediate (like Mg2 in EcoRV,
an endonuclease), serve as bridge between E and
S (holding S in place for catalysis and increase
binding energy, e.g. in NMP kinases and almost
all enzymes that use ATP as S).
3
Proteases
Important in protein degradation and turnover,
and in specific regulatory processing of
selected enzymes and proteins. Mechanism
hydrolysis by adding H2O to the peptide bond
(although thermodinamically favored, it would be
very slow without E 10-103 years).
The resonance structure (partial double-bond
character) makes peptide bonds planar and
kinetically stable. The C of CO gets protected
against nucleophilic attack. The E should
facilitate this nucleophilic attack.
4
Chymotrypsin
Cleaves the the C-terminus of bulkier,
hydrophobic AAs, like Trp, Tyr,
Phe, Met. Good example of a covalent
catalyst. It applies a powerful nucleophile
(Nu) to attack the unreactive carbonyl C. The Nu
briefly gets covalently attach- ed to the S
during the course of catalysis. What is that
powerful Nu in chymotrypsin? The clue came from
the reac- tion with DIPF that showed one
extraordinarily reactive Ser among the 28 Ser
residues in the E.
5
They used a chromogenic ester as S (proteases
often can hydrolyze esters, too) to monitor E
activity. The reaction obeys M-M kinetics with a
KM of 20 mM and a kcat of 77 s-1. They used the
stopped-flow technique that rapidly mixes E and S
(in 1-2 ms) and is able to detect changes
spectrophotometrically right after this fast
mixing.
This proves that hydrolysis proceeds in 2 steps
the burst phase is because the 1st step is
faster than the 2nd one.
yellow color
6
The 2 steps are explained by the formation of a
covalent E-S intermediate.
fast
slow
(intermediate)
p-NO2-phenol
The 3D structure of chymotrypsin (solved in 1967)
shows 3 polypeptide chains linked by S-Ss. It is
synthesized as a single polypeptide
(chymo- trysinogen) which is activated by
proteolysis that provides the 3 chains.
7
The active site
S
catalytic triad
general base catalyst (strong Nu)
Roles His positions Ser and polarize its OH
that it would get deprotonated easier. Asp
orients His and makes it a better H-acceptor
through H-bonding and electrostatic
effects. These observations suggest a mechanism
for peptide hydrolysis
8
protein N-Hs stabilize the negative charge and
the TS
S
(unstable)
mixture of acid-base and covalent catalysis
9
The mechanism does not account for the cleavage
preference of the E. 3D structure with
inhibitors and S analogs revealed a deep
hydrophobic pocket, called S1 pocket, into which
the long, uncharged side-chains of e.g. Phe or
Trp can just fit. Binding an appropriate side
chain into the pocket positions the adjacent
peptide bond into the AS for cleavage.
10
Other proteases have more complex specificity
patterns
Similar ASs and mechanisms are found in homolog
Es (e.g. trypsin, elastase). There is 40
sequence identity and very similar 3D structures
among the- se enzymes. However, their S
specificities are very different trypsin
cleaves after Lys or Arg, elastase cleaves after
Ala or Ser. Looking at the S1 pockets we see
subtle structural differences.
11
Other members of the chymotrypsin family include
proteins involved in blood clotting, the tumor
marker protein prostate specific antigen (PSA),
and many proteases from bacteria, viruses and
plants. There are enzymes having very similar
ASs but they are not homologs of chymotrypsin
(convergent evolution). E.g. bacterial protease
subtilisins AS contains the catalytic triad and
the oxyanion hole, too. Only difference is an
N-H coming not from the backbone but an Asn.
Subtilisin is a founding member of another large
family of proteases.
12
Another example in carboxypeptidase II from
wheat, the structure of which is dissimilar to
the previous 2 Es.
oxyanion hole
Member of an E family with Ac-choline esterase
and certain lipases. All these use His-activated
Nu, but sometimes Cys instead of Ser in the AS.
They also found proteases with Ser or Thr active
groups but activated by NH2 of Lys or the
N-terminus. The AS, mechanistic details and the
roles of nearby AAs can be dissected by
site-directed mutagenesis studies (as long as 1
AA does not change the whole folding of E).
13
How can we be sure that the proposed mechanism is
correct? One way is to identify the individual
contributions to catalytic power of each AA near
the AS using site-directed mutagenesis. Subtilisi
ns AS has been probed by Ala-replacement using a
model S.
Ser-His pair generates the powerful Nu that acts.
N155G mutation (elimina- tion of N-H in the
oxy- anion hole) kcat redu- ced 500X, KM
increased 2X. This proves that N-H stabilizes TS
and then the tetrahedral intermediate. There are
other strategies to cleave peptide bonds Cys-,
Asp- and metallopro- teases.
KM (S binding) unchanged
14
from papaya
Mammalian homologs cathepsins (role in immune
system) Also similar AS caspases (roles in
apoptosis, structure is diffe- rent)
15
approximate 2-fold symmetry
role in regulation of blood pressure
other member of class pepsin (digestive enzyme)
16
closed after S binding
HIV protease, a dimeric aspartyl
protease (cleaves multidomain viral proteins to
their active forms blocking this E blocks the
virus completely to be infectious)
17
member of matrix metalloproteases that remodel
and degrade tissues
base (e.g. glutamate)
other example carboxy- peptidase A (digestive E)
18

• To sum up, all these proteases do 3 things
• Activate a water molecule or another Nu
• Polarize the peptide carbonyl group
• 3. Stabilize the tetrahedral intermediate

19
Protease inhibitors are important drugs
Captopril regulates blood pressure, inhibits the
angiotensin-converting E (ACE), a
metalloprotease. Indinavir (Crixivan), Retrovir
and 20 other compounds treat AIDS, inhibit HIV
protease. To prevent unwanted side effects
these inhibitors must be specific to their
targets not to block other proteases in the body.
Indinavir resembles the peptide S of HIV
protease. It is an alcohol that mimics the
tetrahedral intermediate other groups bind to
the S2, S1, S1 and S2 pockets.
X-ray studies showed that the drug adopts a
conformation in the AS that approximates the
2-fold symmetry of the E. The flexible flaps
fold down on the bound I. The central OH
interacts with the 2 AS Asp. Also, 2 CO on I
are H-bonded to a H2O which is further H-bonded
to N-Hs in the flexible flaps. These
interactions do not form e.g. with renin
(indinavir is specific for the HIV protease).