Download 1 - UCSB C.L.A.S.

CHEM 109C – Zhang
Catalysis II: Enzyme Catalyzed Reactions - KEY
CLAS, F10
1. Label the types of catalysis occurring in the mechanistic step(s) shown below.
Identify the enzyme.
a. Aldolase (gen. B cat. cleavage of the C-3, C-4 bond and gen. A cat.
enamine to imine rearrangement)
b. Lysozyme (Asp 52 acts as Nuc cat. & Glu 35 acts as gen. A cat., Glu 35
can then go on to act as a gen. B cat.)
c. Glucose-6-phosphate isomerase (gen. B cat. increases Nuc of O and
gen. A cat. inc Elec of carbonyl C).
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CHEM 109C – Zhang
Catalysis II: Enzyme Catalyzed Reactions - KEY
CLAS, F10
d. Carboxypeptidase A (Glu 270 as a gen. B cat. and then as a gen. A
cat.)
e. Chymotrypsin (Ser 195 is Nuc cat (inc by gen. B cat. His 57) and His
57 is gen. A cat.)
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CHEM 109C – Zhang
Catalysis II: Enzyme Catalyzed Reactions - KEY
CLAS, F10
2. (Ch 23, #20) If O-18 labeled water was used to hydrolyze lysosyme, which ring
would contain the label?
Hydrolysis occurs in the step shown below and the labeled oxygen would end
up on the anomeric C of the NAM (D) unit.
3. (Ch 23, #23) The descending leg of the pH-rate profile for glucose-6-phosphate
isomerase indicates that one of the amino acid side chains at the active site has a
pKa of 9.3. Identify the amino acid side chain.
With a pKa of 9.3 we look at AAs with side chains that have similar pKas:
Lys (10.79), Cys (8.35), Tyr (10.07)
Since that is the pKa of the group on the descending leg (decreased activity
with increase in pH, more basic soln) we would expect the AA to act as an
acid catalyst (be more active in its protonated/acid form), so it would be Lys.
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CHEM 109C – Zhang
Catalysis II: Enzyme Catalyzed Reactions - KEY
CLAS, F10
4. Below are pH-rate profiles for trypsin and pepsin (digestive protease found in the
stomach). Explain which amino acid residues you would expect at the active site
of each.
Trypsin, with an optimum pH at 6-7, will have AA residues at its active site
that can be protonated and deprotonated at this pH as so we would expect His.
We also know there is Asp and Ser.
Pepsin, with an optimum pH at 2-3, will have AA residues at its active site
that can be protonated and deprotonated at this pH and so we would expect Asp
and Glu residues.
Summary of Enzyme Catalyzed Reactions
1. Carboxypeptidase A
Hydrolysis of the C-terminal peptide bond, release of the C-terminal AA from a
peptide chain.
Metalloenzyme containing Zn2+ (complexed to Glu 72, His 196, His 69, water,
carbonyl O of N-side of C-term AA)
Mechanism
1. Substrate (peptide) binds to active side w/ C-term AA residue in hydrophobic
pocket (therefore NOT good if Arg & Lys are at C-term).
a. Arg 145 & Tyr 248 H-bond (3 of them) to carbonyl and carboxyl Os of
carboxylic acid.
b. Zn2+ complexes & Arg 127 H-bonds (1) to carbonyl O of amide.
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CHEM 109C – Zhang
Catalysis II: Enzyme Catalyzed Reactions - KEY
CLAS, F10
2. Deprotonated Glu 270 acts as a gen. B cat., inc. Nuc of water complexed to
Zn2+, water attacks carbonyl C of amide.
2
C-terminal AA residue is circled in red
Rest of the peptide chain is boxed in blue
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CHEM 109C – Zhang
Catalysis II: Enzyme Catalyzed Reactions - KEY
CLAS, F10
3. Protonated Glu 270 acts as a gen. A cat., inc. LG tendency of amino group &
collapse of carbonyl to form a carboxylic acid (new C-terminus of the
remaining peptide chain).
3
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CHEM 109C – Zhang
Catalysis II: Enzyme Catalyzed Reactions - KEY
CLAS, F10
4. Proton transfer between C-terminal AA residue and new C-terminal group of
remainder of peptide chain.
4
5. Dissociation of C-terminal AA residue and remainder of the peptide chain
from the active site.
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CHEM 109C – Zhang
Catalysis II: Enzyme Catalyzed Reactions - KEY
CLAS, F10
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2. Serine Proteases (trypsin, chymotrypsin & elastase) – Ex. Chymotrypsin
Selectively hydrolyze peptide bonds.
For chymotrypsin
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CHEM 109C – Zhang
Catalysis II: Enzyme Catalyzed Reactions - KEY
CLAS, F10
Called serine proteases b/c all contain Ser, Asp & His (catalytic triad) at active
site.
They differ in the AAs in their binding pockets.
Trypsin: polar, (-) AA residues: Asp 189, Ser 190
– cleaves C-side of Arg & Lys, (+) charged AA residues
Chymotrypsin: small, nonpolar AA residues: Gly 216 & 226
– cleaves C-side of Phe, Trp, Tyr, aromatic AA residues
Elastase: bulky, nonpolar AA residues: Val 216 & Thr 226
– cleaves C-side of Gly & Ala, small AA residues
Mechanism for chymotrypsin
1. substrate (peptide) binds in active site such that
a. appropriate AA residue fits in binding pocket
b. the amide bond to be cleaved is close to Ser 195
2. His 57 acts as gen. B cat., inc Nuc of Ser to attack carbonyl C – Nuc cat.
a. Asp 102 stabilizes the (+) on His 57 and positions the histidine ring w/
basic N towards the OH of Ser 195 – Electrostatic catalysis!
2
Catalytic Triad
N-side
C-side
Phe
3. (-) O of tetrahedral intermediate slips into “oxyanion” hole where it can Hbond to Gly 193 and amine group of Ser 195 (2 H-bonds).
4. Tetrahedral intermediate collapses, C-side of peptide chain leaves.
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CHEM 109C – Zhang
Catalysis II: Enzyme Catalyzed Reactions - KEY
CLAS, F10
a. His 57 acts as a gen. A cat., inc. leaving tendency of amine group
attached to peptide chain (C-side of AA residue in binding pocket).
b. Ser of the enzyme has been acylated – acyl-enzyme intermediate!
3&4
5. His 57 acts as a gen. B cat., inc. Nuc of water to attack carbonyl C of acylenzyme intermediate
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CHEM 109C – Zhang
Catalysis II: Enzyme Catalyzed Reactions - KEY
CLAS, F10
5
6. Tetrahedral intermediate collapses
a. His 57 acts as a gen. A cat., protonating Ser 195 and improving its
“leaving” tendency
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CHEM 109C – Zhang
Catalysis II: Enzyme Catalyzed Reactions - KEY
CLAS, F10
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7. Dissociation of 2 pieces of the peptide chain from the active site (and binding
pocket).
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CHEM 109C – Zhang
Catalysis II: Enzyme Catalyzed Reactions - KEY
CLAS, F10
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3. Lysozyme
Hydrolysis of NAM-NAG bonds/-1,4’-glycosidic bonds
NAM = N-acetylmuramic acid
NAG = N-acetylglucosamine
Overall reaction
@ active site: Glu 35 & Asp 52
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CHEM 109C – Zhang
Catalysis II: Enzyme Catalyzed Reactions - KEY
CLAS, F10
Mechanism
1. Substrate (NAM-NAG polymer chain) binds in enzyme via H-bonding such
that the glycosidic bond between NAM-NAG is in the active site. There is a
change in conformation of the NAM unit from a chair to ½ chair form.
Approximate active site is circled in green
2. Asp 52 acts as a Nuc. cat. and attacks the anomeric C, C-1 of the NAM unit;
Glu 35 acts ac a gen. A cat. and protonates the leaving group on the NAG
unit.
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CHEM 109C – Zhang
Catalysis II: Enzyme Catalyzed Reactions - KEY
CLAS, F10
2
3. LG leaves & Glu 35 acts as a gen. B cat. to inc. the Nuc of water and water
attacks the anomeric C attached to Asp 52.
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CHEM 109C – Zhang
Catalysis II: Enzyme Catalyzed Reactions - KEY
CLAS, F10
3
4. Products dissociate from enzyme
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CHEM 109C – Zhang
Catalysis II: Enzyme Catalyzed Reactions - KEY
CLAS, F10
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4. glucose-6-phosphate isomerase
Converts an aldose to a ketose
@ active site: 1 A cat (Lys?) & 2 B cat (His & Glu?)
Mechanism
1. Substrate (glucose-6-phosphate) binds to active site.
2. Pyranose ring is opened
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CHEM 109C – Zhang
Catalysis II: Enzyme Catalyzed Reactions - KEY
CLAS, F10
a. Gen. B cat. 1 removes proton from hydroxyl group at
anomeric C to reform the aldehyde
b. Gen A cat. protonates the ring oxygen (LG)
2
2
1
3. Gen. B cat. 2 removes a proton from the a-C of the aldehyde & the A
cat. (conjugate A of B cat. 1) protonates the carbonyl oxygen to form
an enol.
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CHEM 109C – Zhang
Catalysis II: Enzyme Catalyzed Reactions - KEY
CLAS, F10
3
Conjugate B of A cat.
2
Conjugate A of B
4. The enol tautomerizes to form acat.1
ketone (see Ch 18).
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CHEM 109C – Zhang
Catalysis II: Enzyme Catalyzed Reactions - KEY
CLAS, F10
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5. Gen. B cat. (conjugate B of A cat.) removes a proton from the oxygen
that got protonated in step 2b which catalyzes the ring closure.
5
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CHEM 109C – Zhang
Catalysis II: Enzyme Catalyzed Reactions - KEY
CLAS, F10
6. Formation of ketal and dissociation of products from active site.
6
5. Aldolase
Cleavage of D-fructose-1,6-diphosphate into D-glyceraldehyde-3-phosphate
(3 Cs) and dihydroxylacetone phosphate (3 Cs)
@ active site: Lys & Tyr
Mechanism
1. Substrate (D-fructose-1,6-diphosphate) binds at active site and forms and
imine bond with Lys
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CHEM 109C – Zhang
Catalysis II: Enzyme Catalyzed Reactions - KEY
CLAS, F10
1
2. Tyr acts as a gen. B cat. to cleave the C-C bond between C-3 & C-4 of the
substrate.
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CHEM 109C – Zhang
Catalysis II: Enzyme Catalyzed Reactions - KEY
CLAS, F10
2
3. D-glyceraldehyde-3-phosphate dissociates & Tyr acts as a gen. A cat. for the
enamine to rearrange to an imine.
3
→
4. Hydrolysis of the imine releases dihydroxylacetone phosphate
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CHEM 109C – Zhang
Catalysis II: Enzyme Catalyzed Reactions - KEY
CLAS, F10
4
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