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3/19/10
1
MANICKAM SUGUMARAN PROFESSOR OF BIOLOGY
U. MASS - BOSTON BOSTON, MA 02125
INHIBITORS - DIFFERENT TYPES
INHIBITORS
REVERSIBLE IRREVERSIBLE
COMPETITIVE
NONCOMPETITIVE
UNCOMPETITIVE COVALENT TIGHT BINDING
AFFINITY LABELSACTIVE SITE REAGENTSPHOTOAFFINITY LABELS
SUICIDAL INACTIVATORS
TRANSITION STATE ANALOGS
SPECIFICNONSPECIFIC
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Active site titration of trypsin
O NO2HN
OH2NNH
HO NO2
FAST
p - Nitrophenyl - p' - guanidinobenzoate (NPGB)
Trypsin
HN
OH
OH2NNH SLOW
HO
HOHN
OH2NNH
O
COMPETITIVE UNCOMPETITIVE NONCOMPETITIVE
REVERSIBLEINHIBITORS
THREE DIFFERENT REVERSIBLE INHIBITORS
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E S
I
+
I
E
E S + P1
P2
Enzyme Substrate
E
Products
Enzyme - inhibitor complex
E-S Complex
Competitive Inhibition Both Substrate and Inhibitor compete for the
same active site on the enzyme
H
HO
O
O
O
O
O
O
O
OO
O
O
Succinate Fumarate malonate
Competitive Inhibitor
Competitive Inhibition
1/v
1/S
No I
[I]"[I]'
[I]"'
0
E + S ES E + P
EI I
Both S and I compete for the same active site on the Enzyme
Slope changes Intercept on Y-axis
no change
At very high [S] conc There is no inhibition
H
HO
O
O
O
O
O
O
O
OO
O
O
Succinate Fumarate malonate
Malonate is a competitive inhibitor of succinate dehydrogenase reaction
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E + E S + P1
P2 Enzyme Substrate
E
Products
Enzyme - Substrate Inhibitor - Complex
E-S Complex
Uncompetitive Inhibition Substrate binds to free enzyme.
Inhibitor binds only to ES complex.
E S
S
I
I
Uncompetitive Inhibitor E + S ES E + P
ESI I
S reacts with E and I reacts Only with ES complex
Slope: No change Intercept on Y-axis:
Varies
Constant amount of ES is removed by I. Therefore, slope is
constant
Uncompetitive Inhibition
1/v
1/S
No I
[I]"[I]'
[I]"'
0
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E + E S +
E P1
P2 Enzyme Substrate
Products
Enzyme - Substrate Inhibitor - Complex
E-S Complex
Noncompetitive Inhibition - Substrate binds to E and EI complex. Inhibitor binds to E and ES complex.
E S
S
E
Enzyme Inhibitor - Complex
S
I
I
I I
Noncompetitive Inhibitor
Noncompetitive Inhibition
1/v
1/S
No I[I]'[I]"
[I]"'
0
Both Slope and Intercept on Y-axis:
Varies
S reacts with E and EI complex. I reacts with E and ES complex.
E + S ES E + P
ESI I
EI I
S
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IRREVERSIBLE INHIBITORS ARE OF TWO TYPES
IRREVERSIBLE INHIBITORS
TIGHT BINDING COVALENT
How to distinguish between reversible and irreversible inhibitors?
TIME
100
0
FOR REVERSIBLE INHIBITOR
FOR IRREVERSIBLE INHIBITOR
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Tight Binding Inhibitors - Transition State Analogs
A-B + C
A--B--C
A + B-C
Reaction Coordinate
Transition State
SubstratesProducts
Transition State Analogs are Potent Enzyme Inhibitors
- Linus Pauling (1946)
Compounds resembling the transition state of enzyme catalyzed reactions
should be effective inhibitors of enzymes.
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Phosphonates are good analogs of phosphate esters
RO
PO
R'O
O
R PO
R'O
OPhosphate ester Phosphonate
Aspartate transcarbamoylase and its transition state analog
OP
NH COOH
OCOOH
O
O
Transition State
N-Carbamoylaspartate
H2N NH COOH
OCOOH
N-(phosphonacetyl)-L-aspartic acid
OP
O NH2
OO
O
Aspartic acidCarbamoyl phosphate
+ OP
O
OO
ONH2
HN COOH
COOH
H2N COOH
COOH
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ACONITASE REACTION
CITRATE
H HOOC OH
H H
O
O
O
O
ISOCITRATE
H OHOOC H
H H
O
O
O
O
H
O
O
O
O
O
O
CIS - ACONITATE
H OHN
O
OH
H H
O
O
O
O
2-NITROISOCITRATE
H OHC
OO H H
O
O
O
O
TRANSITION STATE
H OHN
O
O
H H
O
O
O
O
STABLE ANALOG
Proline racemase reaction
NCOOH
HHN
H
HCOOH
L-ProlineD-Proline
Pyrrole -2-carboxylic acid
Planar Transition State
N COOHH
N COOHH
N COOHH
-1- Pyrroline-2-carboxylic acid
160 fold higher affinity than substrates
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TRIOSE PHOSPHATE ISOMERASE REACTION
GLU 165
C
C
CH2OPO3
H
H O
O
H
N N
HIS9 5
H
2
O
O
GLU 165O
O
C
C
CH2OPO3
H
H O
O
H
N N
HIS9 5
H
2
GLU 165O
O
C
C
CH2OPO3
H
O
O
H N N
HIS9 5H
2
HCH2OPO3
O
O2
C
C
O
2-PHOSPHO GLYCOLATE
ENEDIOL INTERMEDIATE
DIHYDROXY ACETONE PHOSPHATE
GLYCERALDEHYDE -3-PHOSPHATE
Aldolase reaction
OO
H
PHO OH
O
HOH
OHO
H
PHO OH
O
HO N
HO O
HO
POHHO
O
HOHH
H O
OP
OHHOO
Km = 4 x 10 M-4
Dihydroxyacetone phosphate
Glyceraldehyde-3-phosphate
Aldolase
Fructose-1,6-diphosphate
enediol intermediate
Phosphoglycolhydroxamate Ki = 1 x 10 M-8
Transitionstateanalog
+ HO H
OO
POH
HOO
OHHOHH
OP
OHHOO
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Transition state analog -Adenosine deaminase
H2O
OHOH
OHO
N
N N
NOH
NH3
ADENOSINE INOSINE
OHOH
OHO
HNN
NN
HO HTRANSITION STATE
2'-DEOXYCOFORMYCINOHOH
OHO
HN
N N
NHO H
PURINE HYDRATEKi = 3 x 10 M-13
OHOH
OHO
N
N N
NNH2
Ki = 5 x 10 M-6
DIHYDRO PURINECOMPETITIVE INHIBITOR
OHOH
OHO
HN
N N
NHO NH2
OHOH
OHO
HN
N N
NHH
Km = 3 x 10 M -5
Penicillin is a tight binding inhibitor of peptidyltransferase
NN
O
O
OR
O H
H
D-ala D-ala Peptide
H
N
N
O
O
R
OO
SCH3
CH3
H
O
HO
R
N
CH3
O
O
NH3C
H
R N
ON
S
O
COOCH3
H3C
H
Penicillin
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Penicillin inhibits transpeptidase reaction
NN
O
O
O
O H
H
D-ala D-ala Peptide
Penicillin
HN
N
S
O COO
CH3
CH3R
O
HO peptidyl transferase
NO
O
H
O + D-ala
R'N
H2NO
penta gly peptide
HO+Crosslinked peptide
O
N
S
COO
CH3
CH3
O
NH
O
R
H
Inactive peptidyl transferase N
O
O
HR'
NHN
O
Pepstatin is a potent transition state analog of aspartyl proteases (Hydroxyethylene group inside the peptide)
NN
NN
NOH
O
O
O
OH O
O
OH OH
H
H
H
H
N-acetyl PEPSTATIN - A MICROBIAL PEPTIDE
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Methotrexate binds almost
1000 fold tighter than the
substrate to dihydrofolate
reductase
DIHYDROFOLATE REDUCTASE
N
N
N
N
N
N COOH
H2N
O
HH
H
O COOH
12
34
5 6
78
H
H
H
H
7,8-dihydrofolate
Tetrahydrofolate
N
N
N
N
N
N COOH
H2N
O
HH
O COOH
12
34 5
6
78
H
H
H
N
N
N
N
N
N COOH
H2N
NH2
H
O COOH
CH3
H
Methotrexate (4-amino-N-methyl-folic acid)
CARBOXYPEPTIDASE REACTION
O N PN COO
O
H O O
H
O NN COO
O
H O O
H
H
TRANSITION STATE
TRANSITION STATE ANALOG
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OTHER TRANSITION STATE INHIBITORS
• LYSOZYME - LACTONE • RIBONUCLEASE - URIDINE VANADATE • ISOCITRATE LYASE - 3-NITRO PROPIONATE • RIBULOSE BISPHOSPHATE CARBOXYLASE -
CARBOXY ARABINITOL 1,5-BISPHOSPHATE • GLUTAMINE SYNTHETASE -
METHIONINE SULFOXIMINE • GAMMA -GLUTAMYL CYSTEINYL SYNTHETASE -
S-(n-BUTYL)-HOMOCYSTEINE SULFOXIMINE
THERE ARE TWO KINDS OF COVALENT INHIBITORS
COVALENT INHIBITORS
SPECIFIC INHIBITOR
NONSPECIFIC INHIBITOR
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Nonspecific Covalent Inhibitors They react invariably with any enzyme as long as
a particular reactive amino acid is present at or near the active site. Generally the reaction leads to inactivation of the enzyme.
Best examples are active site reagents.
Serine - organophosphorus compounds Histidine, cysteine and Lysine - Haloacetates, Halomethylketones
Histidine -diethylpyrocarbonate Arginine - 1,2-diketocyclohexane
Cysteine - Thiol reagents, pCMB, DTNB Metal ion containing enzymes - cyanide, azide, carbon monoxide
Carbonyl groups - reduction, hydroxylamine Lysine - anhydrides
Carboxyl groups - esterification.
Modification of arginine
N
H R
OHHN
H R
O
N
H
OH
NH
NH2HN
OO+
HN
H R
ON
H R
OHN
H
OH
OH
HO
NH
NHN
Arginine is modified by 1,2-cyclohexadione (or phenylglyoxal). The vicinyl dihydroxy adduct can be stabilized by borate.
Reversible blocking of Arginine by cyclohexadione. E. L. Smith. Methods in Enzymology 47, 156-161 (1977).
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Carbonyl group modifications
NH2OH
NaCNBH3
N
O
HN
OH
Hydroxylamine
Sodium borohydride
Oxime
Carbonyl group
N
O
HN
R
RNH2 Amine
N
O
HNH
RSchiff's base Imine
CHOH groupNHNH2
NO
O
HNaBH4 N
O
O
H
H HNHN
N
O
H
Phenylhydrazine
Phenylhydrazone
Cysteine modification (DTNB)
HN SH
ONO2S
COOH
O2N SHOOC
DITHIONITROBENZOIC ACID (DTNB)
+
O2N SHHOOC
+HN S
O
NO2S
COOH
Addition of excess DTNB leads to quantitative derivatization of cysteine and stoichiometric liberation of the yellow colored aromatic nitrothiol.
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A number of reagents react with cysteine. Ethyleneimine, NE �M, iodoacetate (as well as iodoacetamide) and p-
hydroxymercuribenzoate
will modify cysteine as shown below .
I
HOO HN S
O HOO
CH2CH2
NH HN SH
O
NO O
COOHHOHg
HN S
O
COOHHg
HN S
O
NH2Iodoacetateethyleneimine
N-ethylmaleimide
p-hydroxymercuribenzoate
HN S
O
NO O
Dithiothreitol reduces disulfides and iodoacetate blocks them
SHSH
HO
HOSS
HO
HO
HN
NH
S S
NH
HNO
O
R
R
HN
NH
SH
O
R
HS
NH
HNO
R+
CYSTINE CYSTEINE
Dithiothreitol
Oxidized dithiothreitol
ICH2COOHHN
NH
SCH2COOH
O
R
HOOCCH2S
NH
HNO
R Carboxymethylated cysteines
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Blocking of lysine with TNBS
N
H R
OHHN
H R
O
+
HN
H R
ON
H R
OHN
H
OH
H2NNO2
SO3H
NO2
O2N
N
H
OH
HN
NO2
NO2
O2N
TNBS (2,4,6-trinitrobenzenesulfonic acid arylates lysines.The product can be quantified at 367 nm.
+ H2SO3
Modification of lysine
N
NH2
OH
R R'
O
H2O
N
N
OH
R R'
NaBH4
R R'
N
NH
OH
H
Carbonyl compound Schiff's Base
Lys
Stable imine
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19
Chymotrypsin inactivation by diisopropylfluorophosphate
N NHO
O
OH
HIS 57ASP 102
SER 195
SER 195
N N HO
O
H
HIS 57ASP 102
SER 195
N N HO
O
H
HIS 57ASP 102
F
ASP 102
HIS 57
O
O
H N N H
SER 195
OP
OO
F
O FO
OP
O
OO
OP
O
O
Inactivation of glyceraldehyde-3-phosphate dehydrogenase by p-chloromercuribenzoate
+ Hg
OO
ClSH
Hg
OO
HCl
S
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Iodoacetamide and Iodoacetic acid react with SH, imidazole and NH2
SH +
S
+
S
SH
ICH2COOH
ICH2CONH2
CH2COOH
CH2CONH2
SPECIFIC COVALENT INHIBITORS
AFFINITY LABELS SUICIDAL
INACTIVATOR PHOTO AFFINITY LABELS
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Affinity Label - TPCK
Top : Normal Chymotrypsin substrate Bottom : p-toluenesulfonylphenylalanylchloromethylketone
Arrow indicates the site of cleavage
NH
ONH
O
H3C
SO
ONH
OCl
H2N COOH
TPCK
NORMALSUBSTRATE
TRIOSE PHOSPHATE ISOMERASE RERACTION -AFFINITY LABELLING
DIHYDROXY ACETONE PHOSPHATE
GLU 165O
O
N N
HIS9 5
HC
C
CH2OPO3
H
H O
O
H
2
HGLU 165
O
O
N N
HIS9 5
H
2
C
C
CH2OPO3
H
O
GLU 165O
O
N N
HIS9 5
H
2
C
C
CH2OPO3
H
H Br
O
GLYCERALDEHYDE-3-PHOSPHATEFORMATION VIA ENEDIOL INTERMEDIATE
ACTIVE SITE LABELING BY 3-BROMOACETOL PHOSPHATE
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Affinity label for dihydro-
folate reductase
N N
N
O
N CH3
SOO
F
H2N
NH2
CH3CH3
H
Active site label
DIHYDROFOLATE REDUCTASE
N
N
N
N
N
N COOH
H2N
O
HH
H
O COOH
12
34
5 6
78
H
H
H
H7,8-dihydrofolate
Tetrahydrofolate
N
N
N
N
N
N COOH
H2N
O
HH
O COOH
12
34 5
6
78
H
H
H
PHOTOAFFINITY LABEL
Attach a photoreactive group such as diazoacetyl group to a substrate analog.
Allow the enzyme bind to the photoaffinity label. Shine light on the complex.
The photolabile group is activated and a reactive intermediate is generated.
The reactive intermediate rapidly inactivates the enzyme by binding to essential amino acids near
near the active site.
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PHOTOAFFINITY LABEL - HOW IT WORKS
N2
RNN
O
R CH2
O
R CH2
O
Nu Nu Nu E E Dead
enzyme hν
Step 1. Photoaffinity label binds to the protein. at a site directed by the substituent group (R).
Step 2. Light is shined on the complex to eliminate the photo labile group.
Step 3. The reactive group generated attaches itself to the nearly nucleophile killing the protein.
6E, 11Z-hexadecadienyl diazoacetate interaction with pheromone binding protein
O
O
O CHN2
O
Nu
N2
O CH
O
B
hν
Enzyme
Enzyme
Inactivated Enzyme
3/19/10
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Mechanism of Suicidal Inactivation
I E S’ + E S’ E
E P’
+
E I Inactivation
Product formation
First report on suicidal inactivation -Bloch’s group
NAC
OH O
NACO
NAC
O
β-hydroxydecanoylthioester dehydraseNAC = SCH2CH2NHCOCH3
NACO
H H
H
NACO
NACO
HIS - ENZYME
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Criteria for suicidal inactivation
• Inhibitor should be unreactive. • It should become reactive only after activation
by enzymatic action. • Conversion of unreactive to reactive inhibitor
should be due to specific catalytic function of the enzyme.
• Nonspecific interaction should be minimum. (a) Nontarget enzyme reaction (b) Escape of reactive intermediate before reaction.
How to identify suicidal inactivators?
• Best way to identify is the characterization of enzyme inhibitor complex by physico- chemical studies.
• But this could be difficult in some cases. So one needs some easy techniques to identify the suicidal inactivators.
3/19/10
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Kinetic experiments can identify suicidal inactivators • The loss of enzyme activity should follow time dependent first
order kinetics at fixed concentration of inhibitor. • (If a reactive inhibitor comes out and then reacts with the enzyme, it will obviously
follow second order kinetics. More over second addition of enzyme to this reaction mixture will cause an increased rate of inactivation due to accumulated inhibitor. Also addition of nucleophiles will reduce the rate of inactivation in this mode).
[I]
Time
Logresidualactivity
1
1[I]
k inact
1/k21KI
Kinetics of inactivation
• The rate of inactivation should follow Michaelis Menten type kinetics.
• Substrate (or competitive inhibitor) should protect the enzyme from inactivation.
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Inactivation Kinetics
• Inactivation Should be irreversible. (Show by dialysis, gel filtration etc., )
• Inactivator should be bound covalently to the enzyme (show with radiolabel or other techniques).
• Stoichiometry of binding should be one to one mole.
• Partitioning between catalysis and inactivation should be determined.
Chymotrypsin is an endopeptidase that cleaves proteins on the carboxyl side of aromatic amino
acids (Phe, Tyr, Trp) and Leu.
NH
O
HN
O
COOHH2N
Cleavage site
BINDING SITE
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Suicidal inactivation of chymotrypsin
O
Cl
OO
O
Cl
O
O
CHYMOTRYPSIN
ACYL ENZYME INTERMEDIATE
ACYL QUINONE METHIDE INTERMEDIATE
ACYL QUINONE METHIDE ADDUCT (DEAD ENZYME)
O
NU
HOO OO
O H
NU
Chymotrpsin Suicidal inactivation by 6- chloropyrone
OCl O
O OOCl O
H
OO OX
OCl OO
Chloropyrone
Chymotrypsin Acyl enzyme intermediate
Acid chloride
Tetrahedralintermediate
Hydrolysis and turnover (14 - 40)
Acylated inactive enzyme
3/19/10
29
Highly specific chymotrypsin inactivator
O OI
OX
O OI
OHX
O OI
OX
O OOX
Chymotrypsin Iodomethyl ketone Acylated enzyme
6-iodomethylene napphthyl tetrahydropyran-2-one (1.7 turnover per inactivation)
Stoichiometric titrant for chymotrypsin
O
N
O
OH
Ser- O
O
NH2
OSer -
O
OSer -
NO
O
H
CO2O
N
O
OH
Ser- O
Isatoic anhydride Anthranilyl enyzme resistant to hydrolysis
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30
β- lactamase inhibitor -Sultamicillin
Sultamicillin
N
S
OO
OO
OO
NH2N
O N
S
OO
H
HH
O
+
Amoxacillin - peptidyl transferase inhibitor
Sulbactam - lactamase suicidal inactivator
NH2N
O N
S
O
OO
H
N
S
O
OO
OO
β- lactamase inhibitor - Sulbactam
Sulbactam
tetrahedral intermediate Lactamase Acyl enzyme adduct
Acyl enzyme adduct
+
N
S
O
OHO
O O
Dead enzyme
O N
O COOH
SO2
N
S
O
OHO
O O
O
O N
O COOH
SO2
X
H2N
COOH
SO2
O
X
O
O
3/19/10
31
β- lactamase inhibitor - Olivanate Both natural and synthetic carbapenems containing
pyrroline ring inactivate lactamase by the following mechanism.
Acyl enzyme adduct
H
lactamase
NCOOHO
SN
O
O3SOH
O
ON
COOH
SN
O
O3SOH
O H
Acyl enzyme adduct
tetrahedral intermediate
NCOOHO
SN
O
O3SOH
OH
ON
COOH
SN
O
O3SOH
O
1 2
Lactamase suicidal substrate One mole of acetyl methylene penicillanic acid kills lactamase
with IC 50 value of 1.4 x 10-9 M.
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32
Cephalosporinase inhibitor based on 3’-exo substituent on cephalosporin hydrolysis
F
Ser O
NS
O
NR
O
COOS ArX
O
H
Nu
Ser O
NS
O
NR
O
COOO
SArX
H
Nu
H
Ser O
NS
O
NR
O
COO FS ArX
O
H
Nu
Ser O
NS
O
NR
O
COO FS ArX
O
H
Nu
Ser O
NS
O
NR
O
COOOH
H
NuS ArX
Allyl sulfoxide functionality is uncovered in the acyl enzyme intermediate allowing 2,3 - sigmotropic rearrangement
Sulfenate ester reacts and inactivates the enzyme
β-glycosidase inhibitor : β - D-galacto pyranosyl-p-nitrophenyltriazine in activated a β- glycosidase by
adding on to the methionine at 500 position.
O
OH
OH
OH
OH
NNN
NO2
H
H2N
NO2O
OH
OH
OH
OH
NN
S
S
+
Enzyme
O
OH
OH
OH
OH
NNN
NO2
H2
S
Protonation
S
O
OH
OH
OH
OHInactivation
Cleavage
3/19/10
33
Inactivation of alanine racemase by o-acetylserine
OCOOH
NH2
OH
N
OH
O
P
B
+
B BH
H
Schiff's BaseEnzyme
OCOOH
N
OH
N
OHP
H
Inactive enzyme
N
OHP
COOH
N
OCOOH
N
O
O
OH
N
OHP
H2N
N
OHP
COOH
N
HN
XCOOH
NH2
H
X = halide alsocould do thesame inactivation.
Inactivation of pyridoxal phosphate containing enzymes-1
N OH
O
P
NH 2 HOOC F
H B
+ N HOOC F
H
N OH
P
B
N HOOC
N OH
P
B H F
Nu
H
Schiff's Base Enzyme
Hydrolysis to pyruvate and further turnover
H B
H
N HOOC
N OH
P
Nu Inactive enzyme
However, in three carbon systems, it is the PALP that gets inactivated
and not the enzyme.
3/19/10
34
Formation of PALP adduct
Inactivation is caused by the reaction of product with PALP
EnzymeN
OH
O
P
+ H2N
XCOOH
NH2
HB
N
OHP
COOH
NH HN
B
HSchiff's Base
N
OHP
H2N
XCOOH
N
H
B H
HN
OHP
H2N
XCOOH
N
B
N
OHP
COOH
NH2N
BH
X
N
OHP
COOH
H2NHN
B
N
OHP
H2N COOH
H HN
B
N
OHP
H2N COOH
H2N
B
Inactivation of D-amino acid oxidase by 1-chloro-1-nitroethane
N
N
NH
N
O
OR H
N
N
NH
N
O
OR
H
NO2
HO
N
N
NH
N
O
OR
H
NO2OH
N
N
NH
N
O
OR
H
O
N
N
NH
N
O
OR
H
NO2ClNO2Cl1-chloro - 1 - nitro ethane
Stable N5- acetylated reduced flavin
Cl-
NO2-
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35
Thymidylate synthetase inactivators
S
HN
NOR
NO
O
O
S
HN
NOR
NO
O
O
S
HN
NOR
NO
O
O
5-nitro dUMP derivative
S
HN
NOR
NNN
HN
NOR
NNN
S
bicyclic triazinyl pyrimidine
Thymidylate synthetase inactivator
Inactive enzyme
S
HN
NOR
O SRSR
S
HN
NOR
O SR
H
5-ethynyl-dUMP
S
HN
NOR
O
S
HN
NOR
O SR
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OHOH
O AdS
R
OH
O AdS
R
O OH
O Ad
O
H
H
OH
O AdOH
OOHOH
O AdOH
H2O
L-Homocysteine
Adenosine
H2N SH
COOH
S-Adenosyl-L-homocysteine (SAH)E-NAD E-NADH +
E-NADHE-NAD
Reaction catalyzed by S-Adenosyl-L-homocysteinase
• Inactivation of S-Adenosyl-L-homocysteinase by 2’-deoxyadenosine
OHOH
O AdOH
Adenosine
H
Ara-adenosine
OH
HOH
O AdOH
H
HOH
O AdO
HH
H
O AdO
H
O
OOH
O
Adenine 2'-deoxyadenosine E-NAD E-NADH
Enzyme is locked up in the E-NADH form and is inactive. Accordingly, ara-adenosine is asuicidal inactivator, whileadenosine is not aninactivator.
Ketosugar +
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37
Inactivation by tight binding product
HO
S
HO
SO
S ENZYME
N N
NNFe
Cytochrome P 450 Side chain cleaving enzyme
NADPHO2
22-Thiacholesterol
DEATH OF A REPAIR ENZYME
S
H
CH3S
HN
N N
N
O
H2N
DNA
N
N N
N
O
H2N
CH3
DNAACTIVE ENZYMEO CH3 G RESIDUE-6-
DEADENZYME G RESIDUE
Enzyme uses a cysteine thiol group to abstract the methyl group. The resultant thioether enzyme is irreversibly inactivated.
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38
A SMART WAY TO MAKE THE ENZYME
G RESIDUE
N
N N
N
O
H2N
CH3
DNAO CH3 G RESIDUE-6-
H
CH3O
O
HN
N N
N
O
H2N
DNA
OO
Methanol Hydrolysis
Instead of using cysteine as the active site amino acid, use aspartic acid (or glutamic acid). Once the carboxyl group abstracts the methyl group, use hydrolytic reaction
to regenerate the enzyme.
Inactivation of 3-hydroxyanthranilate dioxygenase
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39
AZT INHIBITS DNA POLYMERASE BY CHAIN TERMINATION MECHANISM
H2COOP
OO
O
A
N3
H2COOP
OO
O
T
P
C
OH
H2COOP
OO
O P Chain termination
Template Primer
AZT
CH2 OOP
OO
O
G
T
O
CH2 OOP
OO
O
CH2 OOP
OO
O
A
5'
3'
3'
5'
3'
5'
3'
5'
3'
5'
3'
5'
HN
N
OCH3
O
N3
CH2 OHO
3'-azido-3'deoxythymidine (AZT)
AZT gets converted into AZT triphosphate and is
incorporated into the growing chain of DNA. But it lacks the 3’ hydroxyl to
allow further growth of the chain. Hence chain termination occurs.
