Professional Documents
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Molecular Enzymology
Narayan Punekar
nsp@
Evaluation Scheme
Assignments & Problem Sets 20
Mid-Semester Examination 30
End-Semester Examination 50
Cyclomaltodextrin-D
-glucotransferase
D-Glucosidase Amylase
STARCH
Pullulanase
Amylase
Glucoamylase
Glucose isomerase
Glucose Fructose
Digestion of FAT
Lipase
(involved in hydrolysis of triacylglycerols)
Inhibition
(lipase activity - enzyme assay)
Inhibitor Screen
(natural or synthetic chemical libraries)
Lipostatin
(active principle)
O H
Pharma Industry N
S
O N
O H O
O
Penicilloic acid
O H Lactamase
N
S
N
O O
Penicillin G O
Penicillin acylase
O
OH
H2 N
S
+
Phenylacetic acid
N
O O
O
6-Aminopenicillanic acid
O H
N
R S
Semisynthetic
N O Penicillins
O
O O O
Cephalosporins
Immobilized Enzymes
E E E E
E E
E E
E E
E E
E E
E E
E E E E
E E
E E
E E
E E
E E E E E E
E E E E
E E
E E
E E
E E
E E
E E
E E E E
E E
E E
E E
E E
E E E E E E
A
Catalysis Allosteric
site
Specificity S
Regulation I
Enzyme Active
site
Cofactor tuning
Redox potential Fe, Heme, FAD
JM Reiner, 1959
enzyme
meaning in yeast in Greek)
(
Ligases, 151
Isomerases,
Oxido-
180
reductases,
Lyases, 462 1295
Hydrolases,
1476
Transferases,
1302
EC nomenclature: Representative classes
4. LYASES: Elimination of a group with double bond formation or addition of group to double bond
4.1.1.1 Pyruvate carboxy-lyase Pyruvate Pyruvate Acetaldehyde+CO2
decarboxylase
TS
Kinetic Barrier: Catalyst
Thermodynamic Barrier: Catalyst
G
+ Energy
uncat
E nergy
G
cat
R e a c t io n c o o r d in a t e
G=H
G H-TS
S and GF=G
G GR
Enzyme only hastens the approach to
equilibrium:
G unaffected
G
(Free
energy)
Equilibrium
Equilibrium
Glucose
Fructose
Glucose
Concentration
Concentration
Fructose
Sucrose
0 0
Time Time
Series of binary collisions
A X
k2>>k1 k!>>k2
A
G
X
P
k1 k2
A X P
Reaction coordinate
Intermediates may accumulate!
[A]0
P
A
k1 k2
A X P
0
Time
Time scales in catalysis
Catalytic turnover
Enzyme binds ligands Covalent modifications
G uncat
E nergy
G cat
R e a c tio n c o o r d in a te
Where:
x is Guncat = -RT ln Keq
y is Gcat = -RT ln Keq
z is GRxn = -RT ln Keq
________________________________________________________________________
Arrhenius Theory TS Theory
k = A e-Ea/RT k = [kT/h] e-G /RT
________________________________________________________________________
k = A e-Ea/RT
k/A = e-Ea/RT
ln (k/A) = -Ea/RT
ln (A/k) = Ea/RT
Guncat = (RT ln 10) log [(kT/h)/k uncat]
Gcat = (RT ln 10) log [(kT/h)/k cat]
-Gcat /RT
k cat = [kT/h] e
G
k uncat = [kT/h] e- uncat /RT
(Guncat-Gcat)/RT
k cat/k uncat = 10
Example: If Guncat = 25.7 kcal/mole and
Gcat = 11.0 kcal/mole
Then,
= 5 x 1010
Origins of Enzyme Catalytic Power
Proximity and
Orientation Effects
Stabilization of
Transition State or Electrostatics
Reactive Intermediate
Enzyme Catalytic
G
)
Power (
General Acid-Base
Metal ion Recruitment
Catalysis
Covalent Catalysis
(Nucleophilic or
Electrophilic)
1.Proximity and Orientation
(Circe Effect)
Carboxylate assisted hydrolysis of phenyl esters and corresponding rate constants
O O O
C C C
H3C O O O
+ O
O
H3C O
C O O
O
NO 2 NO2
O O O O
C C C C
H3C O O O O
H3C
+ + N
N CH3 versus
H3C CH3 versus N
N CH3 NH
CH3 NH
Conformational flexibility and enzyme catalysis
H57 A C
D102
S195
O H N
N HO S
O 2.7A
S
H57
D102
S195
O H N
N HO
O <2.6A
D
D
B
2
1
O2
S S
1
2
LBHB
pKa matching
2.Electrostatic Catalysis
(Pre-organized active site)
A C
H3C R2 H3C R2
- -
+
+
R1 N S R1 N S NH2
H3C + CO2
O H3C OH HOOC COOH
HO
O
N155 B
O
O O
N
H N + H2N
E-Ser-O- E-Ser-O H E-Ser-O
(S221) (S221) (S221)
Tetrahedral intermediate
Charge Guidance
AcCh+ O2 -
AcCh+ O 2-
+ O 2- -
AcCh+
-
+
B
O P
O P O P
H2C
H2C H2C
2+
O Zn 2+ 2+
O Zn O Zn
HC
OH R HC HC
OH OH
R
H H C
N N
O HO H O OH
2+ 2+
Zn Zn
H+
N N N
N N N
Enzyme Enzyme
Better nucleophilicity to water
A B
O H
2+
O 2+
C Mn Mn
HO
2+ O
Zn O O
N N
N
Enzyme
Asp
Enzyme
Charge Shielding
Nu -
O O O
O O
O P P P O
N
H2N O O O O
N
N 2+
N Mg
HO OH
A H A H
O O O
O O O
H H H H H H
B B
Acid catalysis Base catalysis Concerted Acid-Base catalysis
Functional group Structural form Typical pKa Acts at
Acid Base (Range) pH 7.0 as
R O R O
Carboxylate 3.4 (2.0-7.0) Base
-COOH (C- OH O 3.9 (2.0-7.0)
terminal) 4.3 (2.0-7.0)
-COOH (Asp)
-COOH (Glu)
H
Imidazole (His) +
R N 6.5 (6.0-8.0) Acid or
R N
Base
N N
H H
-Amino (N- O O 7.5 (6.0-8.0) Acid
+
terminal) NH 3 NH 2
R R
+
-Amino (Lys) NH 3 NH 2 10.5 (6.0-10.5) Acid
R R
SH S
Bronsted acid-base
Sulfhydryl (Cys) 8.3 (7.5-9.0) Acid
R R groups on enzymes
Hydroxyl (Tyr) OH O 10.0 (8.5-10.5) Acid
R R
+
Guanidinium (Arg) NH 2 NH >12.0 Positive
N N
NH 2
Charge
NH 2
R R
Hydroxyl (Ser, Thr) OH O 13.5 (9.0-13.5) Acid (?)
R R
+ + + +
+ + + +
NH3 NH2 NH3 NH2
O OH O O O OH O O
C C C C
O OH O O O OH O O
C C C C
OH
O C D
R1OCH2
O Base
:O [His12]
:N
[Asp52]-COO- O OH
O
HOOC-[Glu35] NH
[His119]
O P O
+
O HN N H OR2
5.Stabilization of Transition State
O A OH B
COOH
OH OH
C H 3C H H 3C
O O O
O HO OH
D N O O
O:
HO H
OH OH HO OH HO OH
NAM HO
C H3C O N H
A c a rb o s e
O
C
E
O S ta tin
O NAG H
N NH A la S ta -C O O H
C H 3C O N H Val Va N
H
O OH O
F
NH2
N
N
O
N N NH A la
O
N
H OH
HO OH OH
HO
A d e n o s in e
OH
D
H
N
O O O
HN C C C
N N N
N N H H O H O H O
O
HO OH P ro lin e TS P y r r o le -2 -c a r b o x y la te
HO
N e b u la rin e 1 ,6 -h y d ra t e E
H H C H 2O P -OOC C H 2O P -OOC C H 2O P
N
O
HN C C OH C OH
N
N OH O OH
O HO HO HO
HO OH
HO
C H 2O P C H 2O P C H 2O P
C a rb o x y k e to n e 2 ' C a r b o x y - D - a r a b in i t o l
1 , 6 - D ih y d r o n e b u l a r i n e R ib u l o s e 1 , 5 - b is p h o s p h a t e
1 , 5 - b is p h o s p h a t e
I think enzymes are molecules
that are complementary in structure
to the activated complexes of the
reactions that they catalyze.
..ABZYMES!!
Where does the G for TS binding
come from?
A B C D E F
--NAG-NAM-NAG-NAM-NAG-NAM--
DHAP
H OH O O OH
C H C H C N
A-H :B
OH OH
OH O
H2C P H2C H3C H2C
O P O P
METHYL GLYOXAL
ENEDIOL Phosphoglycohydroxamate
TS mimic (155 times tighter binding)
GAP
6.Covalent catalysis
Enzymes recruit
Electrophiles (rare on a protein!)
Nucleophiles (many on a protein!)
Examples of covalent reaction intermediates
C ovalently linked to Interm ediate E nzym e exam ples
E n zym e provides th e n ucleoph ile
S erine (-O H ) O -A cyl enz ym e A cetylcholinesterase, C hym otrypsin
C ysteine (-S H ) S -A cyl enzym e P apain, G lyceraldeh yde-3-phosphate
dehydrogenase, G lutam ate synthase
S erine (-O H ) O -P hospho enzym e A lkaline phosphatase,
P hosphoglucom utase;
P hosphodiesterase (via T hr-O H )
H istidine N 1 -P hospho enzym e G lucose-6-phosph atase, N ucleoside-
(-im idazole) bisphosphate kinase, Succinyl C oA
synthetase
T yrosine (-O H ) O -S ulfo enzym e A rylsulfate sulfotransferase
L ysine (-N H 2 ) Im ine adduct (S chiffs Fructose-1,6-bisphosphate aldolase,
base) A cetoacetate d ecarbox ylase,
T ransaldolase
L ysine (-N H 2 ) A M P -enzym e D N A ligase (N A D + )
G lutam ate (-C O O H ?) G lycosyl enzym e S ucrose phosphorylase, -
G alactosidase
E n zym e provides th e electroph ile
P yruvo yl group Im ine adduct (S chiffs H istidine decarbox ylase (bacterial)
base)
P yridoxal phosphate A ldim ine (S chiffs G lutam ate decarbox ylase, L-A lanine
base) am inotransferase
B iotin N -C arbox y-biotin A cetyl C oA carbox ylase,
T ranscarbox ylase, P yruv ate
carbox ylase
T hiazolium ring of H ydrox yeth yl T P P ; P yruvate decarbox ylase;
T hiam ine 1,2-D ihydrox yeth yl T ransketolase
p yrophosphate TPP
Schiff base Lysine NH2
Schiff Base formation
H3C H3C +
CH3
H HA .. N
N O N OH H + OH-
H
H X
X X
B:
H
+
C .. C
N H N
H H
X X
codon usage
Polypeptide Polypeptide
N
H O
COOH
Glutamate + Tyrosine Post- HOOC HN 2
Bacterial glucose
1
O
Serine Pyruvate Post- H
N Pyruvoyl enzymes
H3C Polypeptide
translational; O
like histidine
peptide decarboxylase
bound
CH2
(Alanine Post- Polypeptide Polypeptide Phenylalanine
Dehydroalanine) translational; N
H ammonia lyase
O
-Ala-Ser-Gly- - peptide and histidine
MIO- (4- bound CH2 ammonia lyase
methylideneimidazole- N
O
O
5-one) N
Polypeptide Polypeptide
Coenzyme/ Structure Enzyme example Vitamin
Cofactor (function)
HO
Ascorbic acid O O Prolyl hydroxylase Vitamin C
HO
(Redox)
HO OH
O
Biotin Pyruvate carboxylase Biotin
HN NH
(Electrophile)
COOH
S
H2N N N
Tetrahydrofolate Serine Folic acid
N
(FH4) N hydroxymethyltransferase
OH N
H
NH Glutamate (Redox and electrophile)
O
H H
Nicotinamide CONH2 Lactate dehydrogenase Niacin
O
adenine O
(Redox)
N O P O P O N
dinucleotide O O O O
N
NH2
(NADH and HO OH
N N
HO OH
NADPH) (P)
O
Coenzyme A N O P O
Citrate synthase Pantothenic
O
(CoA) H2N
N
O O
HO
(Acyl activation; good acid
N
N N
HO
O
O P O
H H
N
SH
leaving group)
O O
O
Pyridoxal O
HC GABA transaminase Pyridoxal
OH
phosphate O P O (Electrophile)
O
(PLP) N
O
Flavin adenine HO
HO
O P O
D-Amino acid oxidase Riboflavin
dinucleotide OH
OR
(Redox)
(FAD) and N N O
Flavin NH
N
mononucleotide O
(FMN)
O
Thiamine O
P O
Transketolase Thiamine
O P O
pyrophosphate O
O (Electrophile)
(TPP) N
C
N
+
S
N NH2 H
1. The groups/ atoms relevant to chemistry are marked grey in each structure. 2. NADP+
differs from NAD+ in having a phosphate group on 2 OH (shown as (P)). 3. R= H for FMN and R=
AMP attached via its phosphate for FAD. 4. Other cofactors like lipoic acid, cyanocobalamin
(vitamin B12) and phylloquinone (vitamin K) also perform important functions but are less
Detection of reaction intermediates and
establishing their catalytic competence
Stereo-chemical evidence
A405
[E] = 0.5 mg/ml
Time
O
O O
Fast Slow
NO2 H3C O
H3C O H3C OH
Ser-Enz OH-
O2N O (Acyl enzyme)
Enz-Ser-O- Enz-Ser-O-
(Chymotrypsin) (Chymotrypsin)
Glutamine synthetase reaction A
O
O
+ O
+
O NH3
O NH3 +
AMP O NH3
O ADP Pi
P O :NH3
O O C
O C O O C
O O
P O O
O P O NH2
O
O
L-Glutamate + ATP -Glutamyl phosphate L-Glutamine
18O PIX by glutamine synthetase B
O + AMP O + AMP O + AMP
NH3 O NH3 O NH3 O
O
P
O
P
O
P
Isotope exchange (PIX)
O O
C O
O
C O
O
O
O
C O
O
(Also in Ping-pong cases)
O O O O O
P O P O P O
O O O
O O O
Analog phosphorylation D
O O
+ O + O
H3N H3N
ATP ADP
O
H3C S NH H 3C S N P
O
O O O
L-Methionine- L-Methionine-
S-sulfoximine S-sulfoximine-P
Nucleophile or Base?
Reaction intermediate trapping: An Example
O
Uncatalyzed
O
H3C O NO2 HO NO2 +
O
H 3C OH
H H
O
O Catalyzed Catalyzed O
Step 1 + Step 2
NO2 H3 C N NH
H3C O H3C OH
O +
HN N H HN NH
H
N-Acetyl imidazole
+
O2N O
Acetate catalyzed Hydrolysis of p-Nitrophenol
O O
O O
O H H
H3C O
H3C O
O O
H3C O CH3
(Acetic anhydride)
NH2
H2O
TRAP
NHCOCH3
2 CH3COOH + CH3COOH
Acetanilide
Imidazole catalyzed Hydrolysis of p-Nitrophenol
O O
O
H H
HN N
HN N
O
+
H3C N NH
(N-Acetyl imidazole)
NH2
H2O
TRAP
CH3COOH NHCOCH3
+ HN N + HN N
Acetanilide
Enzyme reaction intermediates trapped by
borohydride reduction
In the first three examples the intermediate is covalently bound to the enzyme.
In the last case, the intermediate is non-covalently held at the active site.
Stereochemical evidence
Retention of stereochemistry at C1 - Covalent glycosyl intermediate
HO H
HO-Sugar
O O-Sugar O O OH
H
C C C
H E-COO H
E-COO- E-COO-
HO-Sugar
O O-Sugar O H
C C
H
OH
E-COO- H OH E-COOH
Steps - Odd
Steps - Even
Identification of catalytic residues
Besides the trapping experiments (for Nu:)
Chemical modification
pH dependence of catalysis
X-Ray and other structural data
Site directed mutations
Chemical modification
Table 3.5 Chemical modification reagents as irreversible enzyme inhibitors
Amino acid (functional group) Commonly used reagents
Arginine (guanidinium) 2,3-Butanedione, Phenylglyoxal
Cysteine (thiol) Iodoacetamide, N-Ethylmaleimide (NEM),
4-Chloromercuribenzoate, Disulfides
Histidine (imidazole) Diethylpyrocarbonate (DEPC)
A B
ko k p
log = n log[ P] log K P
kp
Active site directed irreversible inhibitors
:Nu
Peptide substrate
O R
H
N H C C
C C N H C
H
O H2C O
TPCK
:Nu
H3C
O
H
N H C Cl
S C C
OO HC H2
2
Suicide Substrates (Trojan Horse!)
General Acid-Base
Metal ion Recruitment
Catalysis
Covalent Catalysis
(Nucleophilic or
Electrophilic)
Peptide bond hydrolysis:
Example of which enzyme uses what
O El +
N u: C
N H -X
H
Feature Serine Cysteine Aspartyl Metallo-
protease protease protease protease
1. Elementary processes