enzymes and enzyme kinetics 2012
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ENZYMES
OBJECTIVESAt the end of the lecture, the student should be able to: 1. Define the following: a. Enzymes b. Apoenzyme c. Coenzyme d. Holoenzyme e. Metalloenzyme f. Regulatory enzyme g. Active site of the enzyme h. Allosteric site of the enzyme i. Substrate 2. Discuss the helpers (cofactors) of enzymes.
3. Enumerate the six major classes of enzymes.4. Discuss the characteristics of enzymes. 5. Explain the models of enzyme-substrate complex. 6. Explain enzyme kinetics. a. Factors that affect enzyme activity or
reaction velocity.b. Ways of expressing enzyme activity.
7. Discuss the operation and plots used to illustrate enzyme kinetics.
a. Michaelis-Menten kineticsb. Lineweaver-Burke Double Reciprocal Plotc. Michaelis constant and its significanced. Kinetic order of reactions
OBJECTIVES
8. Discuss enzyme inhibition and its effect on reaction velocity. a. Reversible b. Irreversible9. Discuss the different ways of regulating enzyme activity.10. Explain the factors affecting enzyme activity.11. Elucidate uses and clinical application of
enzymes.
OBJECTIVES
ENZYMES
Specialized protein catalysts that accelerate chemical
reactions
APOENZYME APOENZYME APOENZYME
DEFINITION OF TERMS
Protein part
Cofactor (Nonprotein
part)Coenzyme
Prosthetic group
Metal ion
HOLOENZYME
+ ++
ENZYME COFACTORS
A. Coenzyme EnzymeChemical Groups
Transferred
VitaminPrecursor
Thiamine Pyrophosphate
(TPP)
Pyruvate dehydrogenase, Isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, Transketolase, α-Ketoacid dehydrogenase
Aldehydes Thiamine (Vit B1)
Flavin Adenine Dinucleotide
(FAD)
Succinate dehydrogenase, α-Ketoglutarate dehydrogenase, Pyruvate dehydrogenase, Nitric oxide synthase
ElectronsRiboflavin
(Vit B2)
Nicotinamide Adenine
Dinucleotide (NAD)
Lactate dehydrogenase; Other dehydrogenases
Hydride ion(:H-)
Nicotinic acid(Niacin; B3)
Pyridoxal Phosphate (PLP)
Glycogen phosphorylase, γ-ALA synthase, Histidine decardoxylase, Alanine aminotransferase
Amino groupsPyridoxine
(Vit B6)
Lipoate Pyruvate dehydrogenaseα-Ketoglutarate dehydrogenase
Electrons and acyl groups
Not required in diet
ENZYME COFACTORS
A. Coenzyme EnzymeChemical Groups
Transferred
Vitamin Precursor
Coenzyme A(CoASH)
Acetyl CoA carboxylase Acyl groups
Pantothenic acid & other compounds
Biocytin
Pyruvate carboxylase, Acetyl CoA carboxylase, Propionyl CoA carboxylase
CO2
Biotin
5’-deoxycobalamin
Methylmalonyl mutase
H atoms and alkyl groups
Vit B12
TetrahydrofolalateThmidylate synthase
One-carbon groups
Folic acid
CLASSES OF COENZYMES
CLASS EXAMPLES
Activation-Transfer Coenzymes
TPPCoenzyme A
BiotinPLP
Oxidation-ReductionCoenzymes
NAD+
FADVit E, Vit C
Mark’s Medical Biochemistry, 3rd ed.
ENZYME COFACTORS: COENZYME A
C-CH2-CH2-N-C-C—C-CH2O
OII
OII
IOH
H
IIO
I CH3
CH3
I
I NH
I CH2
I CH2
I SH
O = P – O-
IOI
O = P –O-
IO
NH2
NN
N N
OI
O = P – O-
IO-
O
OH
Pantothenic acid
1. Pantothenic acid-derived, co-factor of several enzymes like acetyl CoA carboxylase.
2. Takes part in reactions of the CAC, FA synthesis and oxidation, acylations and cholesterol synthesis.
H
H
HH H
Active sulfhydryl
group that form thioesters with
acyl groups
COO-
| CH2
| CH2
| C = O |
S ~ CoA
COOH | CH2
| CH2
| C = O | COO-
α-Ketoglutarate (C5) Succinyl CoA (C4)
NAD+ NADH + H+
CO2CoASH
α-Ketoglutarate Dehydrogenase Complex
Coenzymes:1. TPP2. Lipoic Acid3. FAD4. NAD+
5. CoASH
ΔG0 = - 8.0 kcal
ENZYME COFACTORS: COENZYME A
Pyruvate decarboxylaseDihydrolipoyl transacetylaseDihydrolipoyl dehydrogenase
COO-
|C=O|CH3
S ~ CoA |C=O |CH3
NAD+NADH+ +
H+
CoASH
Pyruvate Dehydrogenase Complex
TPP LipoateFAD
Pyruvate decarboxylaseDihydrolipoyl transacetylaseDihydrolipoyl dehydrogenase
Pyruvate(C3)
Acetyl CoA(C2)
CO2
ΔG0 = - 8.0 kcal/mole
ENZYME COFACTORS: COENZYME A
ENZYME COFACTORS: NAD & NADP
OHOH
O II
C – NH2
NIR
O
-O
N
N
NH2
N
N
O
H
O – CH2
HH
O
H
OH
– CH2
HH
OH
O
-O
P
O
O
-O
P
OII
C – NH2
H
N
NADP+ contains a Pon this 2’-hydroxyl
H
H
Nic
oti
na
mid
e a
den
ine
din
uc
leo
tid
e (
NA
D+)
Adeninering
Ribose ring
+
COO-
I
H-O-C-H I
CH3
Lactate
Dissociates as H+
COO-
I C=O + H+
I CH3
Pyruvate
1
Nicotinamidering
CC
Ketogroup
1
3
2
Functionalgroup
2
1
1’
2’
349
87
65
AMP provides additionalbinding interactions that induce conformational changes in the enzyme
AM
P
Lactatedehydrogenase
3’2’
2’
3’
5’
4’
ENZYME COFACTOR: NAD+
COO-
| HO - C – H | CH3
COO-
I C = O
I CH3
Lactate dehydrogenase
L-Lactate PyruvateNAD+ NADH+ + H+
H
H
R
H-N N-H
S
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIH
O
IIIIIIIIIIIIIII
II
H
COENZYME: BIOTIN
CO2
attachment site
NH
NH
CIIO
H
H-N N-H
S
IIIIIIIIIIIIIIIIIIIIIIIIIH
O
IIIIIIIIIII
II
= O
Biotin
Protein portion of enzyme:Acetyl CoA carboxylasePropionyl CoA carboxylasePyruvate carboxylase
Lysyl residue
Biotin
1. One of the B complex vitamins.
2. A cofactor of such enzymes as acetyl
CoA carboxylase, propionyl CoA
carboxylase and pyruvate carboxylase.
3. It is a carrier of activated CO2, hence involved in
carboxylation reactions.
OII
COENZYME: BIOTIN
Pyruvate
Gluconeogenesis
Oxaloacetate
ATP ADP + Pi
Pyruvate carboxylase
CO2BIOTIN
COO-
I C =O
I CH3
COO-
|C= O|CH2
|COO-
COENZYME:THIAMINE PYROPHOSPHATE
H
Thiamine Pyrophosphate(TPP; Vit B1-derived)
N
H3C
NN NC H
HS
C-C-OHH
AMP
CN
NH2
CH
CNO-
IIO
OII
CH2
H3C
NH2
S
C
C
C
N
H
CH3
- CH2 –CH2 – O – P –O – P – O-
O-
Dissociable proton
Reactivecarbon atom:
carrier of aldehyde groups
Coenzyme binding
site
Succinate dehydrogenase
(C6) OXALOACETATE CITRATE (C6)
a – KETOGLUTARATE (C5)
SUCCINYL CoA (C4)
ISOCITRATE (C6)
(C4) FUMARATE
(C4) SUCCINATE
(C4) MALATENAD
NADH + H+
FADH2
FAD
H2O
NAD
NADH + H+
CO2
H2O
H2O
Malate dehydrogenase
Isocitrate dehydrogenase
Succinyl CoA thiokinase
PYRUVATE
ACETYL-CoA
Citrate synthase
NAD
NADH + H+
CO2
Pyruvate
dehydrogenase
(ATP)
GTP ADP + Pi
CoAMg+
NAD
NADH + H+
CO2
Aconitase
Fumarase
a - ketoglutarate dehydrogenase
(C3)
(C2)
ETC
H2O O2
ATPOxid.
Phospho
Role of TPP in the Oxidative Decarbo-
xylation Reactions of CAC
TPP
TPP
TPP
H3C
NH2
O
H3C
H3C
NHN
NN O
CH2
I H-C-OH
I H-C-OH
I H-C-OH
I CH2-O-P PO O-CH2
OII
IO
P
NH2
N
N
N
N
O
OH OH
OII
COENZYME: FAD
Flavin Adenine Dinucleotide (FAD)
A riboflavin (Vit B3)-derived coenzyme of several dehydrogenases involved in
oxidation-reduction reactions.
Isoalloxacin ring
Ribitol
From ATP
COOH |
CH2
| CH2
| COOH
H -COOH | H – C ||
C – H |
H -COOH
Fumarate (C4)Succinate (C4)FAD FADH2
Succinate dehydrogenase
ROLE OF FAD IN THE CITRIC ACID CYCLE
COENZYME: FAD
ROLE OF FAD AND FMN IN NITRIC OXIDE (NO) SYNTHESIS
Arginine
NO +
NADPH2
+ O2
NADP+
+ H2O
Nitric oxide synthaseFAD, FMN, HemeTetrahydrobiopterin
COENZYME: FAD NH2
I H2N = C I
NH I
CH2
I CH2
I CH2
I H3N – C – H
I COO-
NH2
I C = O
I NH
I CH2
I CH2
I CH2
I H3N – C – H
I COO-
+
Citrulline
+
+
-O3PO-CH2
CH3
C HO
N
H
OH
OH
Pyridoxal Phosphate
COENZYME:PLP
A Vit. B6-derived coenzyme involved in carbohydrate, amino acid and neurotransmitter synthesis.
+
Reactive aldehyde groupinvolved in the transfer
of amino groups.
ROLE OF PLP IN CARBOHYDRATE METABOLISM: GLYCOGENOLYSIS
O
OH
OH
HO
O-PO3=
O
OH
OH
HOO
OH
OH
HO
O
O
OH
OH
HO
O
O
OH
OH
HO
O
Glycogen chain
Glygogenphosphorylase
Pi
O
OH
OH
HO
O
O
OH
OH
HO
O
OH
OH
HO
O+
OH
OH
OHH
OH
Glucose 1-PRemaining glycogen
PLP
H
H
H
H
H
H H
H
H
H
H
H
COENZYME: PLP
ROLE OF PLP AS A COENZYME IN AMINOACID METABOLISM: HEME SYNTHESIS
Glycine +Succinyl CoA
δ-Aminolevulenic acid(ALA)
Heme(Fe protoporphyrin IX)
several reactions
δ-Aminolevulenate synthase PLP
COENZYME: PLP
ROLE OF PLP IN AMINO ACID METABOLISM: HISTAMINE SYNTHESIS
H|
CH2 – C – COO-
| NH3
PLP
CH2 – CH2 – NH3
Histidine
HISTAMINE
CO2
Histidinedecarboxylase
COENZYME: PLP
NH3+
| CH3 – C – C – COO-
|H
Alanine
COO-
| CH2
| CH2
| H– C – NH3
+
| COO-
Glutamate
ROLE OF PLP IN AMINO ACID METABOLISM:TRANSAMINATION
O||
CH3 – C – COO-
Pyruvate
COO-
| CH2
| CH2
| C = O
| COO-
α-Ketoglutarate
+ +
Alanine aminotransferase
(transaminase)
PLP
COENZYME: PLP
Cofactor EnzymeB. Inorganic (Metal ions or iron- sulfur clusters)
Zn+2 Carbonic anhydrase, Alcohol dehydroge- nase, Carboxypeptidases A & B
Cu+2 Cytochrome oxidase
Mn+2 Arginase, Ribonucleotide reductase
Mg+2 Hexokinase, Pyruvate kinase, Glucose 6- phosphatase
Ni+2 Urease
Mo Nitrate reductase
Se Glutathione peroxidase
Mn+2 Superoxide dismutase
K+ Propionyl CoA carboxylase
ENZYME COFACTORS
ENZYME COFACTORS: Mg+2
Glucose Glucose 6-PO4
HexokinaseGlucokinase
ATP ADP + Pi
Mg+2
O || C1 - H | H - C2 - OH |OH - C3 - H | H - C4 - OH | H - C5 - OH | H - C6 – OH | H
O || C1 - H | H - C2 - OH |OH - C3 - H | H - C4 - OH | H - C5 - OH | H - C6 - O – P | H
GLYCOLYSIS
COO-
| C2 = O | CH3
O || C1 – O-
| C2 – O ~ P |H – C3
| H
Phosphoenol Pyruvate(PEP)
PyruvateADP ATP
Mg+2
K+
Δ G0 = - 6.1 kcal/mole
ENZYME COFACTORS: K+
Pyruvate kinase
ENZYME COFACTORS: Zn+
CO2 + H2O H2CO3
Carbonic anhydrase
Zn+2
METALLOENZYMES
Enzymes that requirea metal in their
composition
SUBSTRATE
The molecule acted upon
by the enzyme
to form a
product
Hexokinase/Glucokinase
ATP ADP
Mg+2
Δ G0 = - 4.0 kcal/mole
O || C1 - H | H - C2 - OH |OH - C3 - H | H - C4 - OH | H - C5 - OH | H - C6 – OH | H
Glucose
O || C1 - H | H - C2 - OH |OH - C3 - H | H - C4 - OH | H - C5 - OH |
H - C6 - O – P | H
Glucose 6-Phosphate
ATP AS A CO-SUBSTRATE
ACTIVE SITE OF THE ENZYME
LYSOZOME: ACTIVE SITE
CHYMOTRYPSIN:ACTIVE SITE
His 57 Ser 195
ALLOSTERIC SITE
Substrate
Enzyme
Allosteric site
Substrate sites
REGULATORY ENZYME
The enzyme that catalyzes therate-limiting or committed
step of a metabolicpathway.
REGULATORY ENZYME Phosphofructokinase I
Fructose 6-phosphate
Fructose 1,6-bisphosphate
ATP ADP + Pi
Phosphofructokinase I
Glycolysis
H | H - C1 - OH | C2 = O |OH - C3 - H | H - C4 - OH | H - C5 - OH | H - C6 - O – P | H
H | H - C1 - O - P | C2 = O |OH - C3 - H | H - C4 - OH | H - C5 - OH | H - C6 - O – P | H
AMPF 2,6 bisPO4
ATPCitrateH+
+ -
O ||CH3 – C – S – CoA
ACETYL CoA
ATP
ADP + Pi
O O \ || C – CH2 – C – S – CoA //O
MALONYL CoA
Acetyl CoA carboxylase
CO2
(HCO3-)
CitrateInsulinHigh CHOLow FatHigh Prot.
Malonyl CoAPalmitoyl CoAEpinephrineGlucagonHigh FatFasting
H2O
+
-
REGULATORY ENZYMEAcetyl CoA Carboxylase
De Novo Synthesis of Fatty Acids
HMG CoA Mevalonate
HMG CoA reductase
NADPH + H+
NADPH
CoA
REGULATORY ENZYMEHMG Coa Reductase
Cholesterol Synthesis
C1OO-
| C2H2
| HO – C3 – CH3
| C4H2
| C5H2OH
O II
-O – C I
CH2
I OH –C – CH2
I C – S – CoA
II O
Insulin, T3Glucocorticoids
Cholesterol GlucagonBile acids StatinsMevalonate
-+
Glucose 6-phosphate dehydrogenase
NADP NADPH + H+
Pentose Phosphate Pathway
REGULATORY ENZYMEGlucose 6-Phosphate
Dehydrogenase H |
C1 = O |
H – C2 – OH |
HO – C 3– H |
H – C4 – OH |
H – C 5– OH |
C6H2OPO32-
O ||
C1 |
H – C2 – OH |
HO – C3 – H |
H – C4 – OH | H – C5
|
C6H2OPO32-
Glucose 6-phosphate 6-phosphogluconolactone
INTRACELLULAR LOCATION OF SOME IMPORTANT BIOCHEMICAL PATHWAYS
ISOENZYME
Different structural forms of an enzyme which catalyze the same chemical reactions → act on the same substrate(s) and produce the same product(s) but exhibit differing degrees of efficiency.
Different isoenzymes are expressed in specific tissues of the body.
ISOENZYMES OF LACTATE DEHYDROGENASE
Lactate dehydrogenase (LDH) – catalyzes the reversible conversion of pyruvate to lactate.
Tetramer consisting of 2 subunits: M (found in skeletal muscles and
liver) & H (heart).5 distinct isoenzyme forms (from combination of M & H isozymes).
An increase of H4 in the blood indicates tissue damage as in heart attack.
Enzymes can therefore serve as markers for disease.
SIX MAJOR CLASSES OF ENZYMES (IUBMB*, 1964)CLASS EXAMPLE
Oxidoreductases Dehydrogenases, Oxidases, Reductases, Peroxidases, Catalases, Oxygenases, Hydroxylases
Transferases Transaldolase and Transketolase; Acyl, methyl glucosyl, and phosphoryltransferases,Kinases, Phosphomutases, Transaminases
Hydrolases Esterases, Glycosidases, Peptidases, Phosphatases, Thiolases, Phospholipases, Amidases, Deaminases, Ribonucleases
Lyases Decarboxylases, Aldolases, Hydratases, Dehydratases, Synthases, Lyases
Isomerases Epimerases, Isomerases, Mutases, Racemases
Ligases Synthetases, Carboxylases
*International Union of Biochemistry and Molecular Biology; classification is based on the reactions enzymes catalyze; each class is divided into subclasses.
OXIDOREDUCTASES
Transfer of electrons and hydrogen atoms from donors (or reductants,
hence oxidized to acceptors (or oxidants, hence reduced).
COO-
| HO – C – H + NAD+
| CH3
L-Lactate
COO-
| C = O + NADH + H+
| CH3
Pyruvate
Lactate dehydrogenase
TRANSFERASESTransfer functional groups (like C-, N-, or
P-) from donors to acceptors; utilize 2 substrates to produce 2 products.
COO-
| H3N – C – H + C = O | | CH3 (CH2)2
L-Alanine | COO- α-Ketoglutarate
(keto acid)
COO- COO-
| | C = O + H3N – C – O | | CH3 (CH2)2
Pyruvate | COO-
L-Glutamate (amino acid)
Alaninetransaminase
PLP
(amino acid) (keto acid)substrate
substrate
product
product
VI. Reactions: GLYCOLYSIS Kinase - transfers the functional group phosphate from ATP to an acceptor
ATP(donor)
ADP + Pi(product)
Mg+2
Δ G0 = - 4.0 kcal/mole
O || C1 - H | H - C2 - OH |OH - C3 - H | H - C4 - OH | H - C5 - OH | H - C6 – OH | H
Glucose(acceptor)
O || C1 - H | H - C2 - OH |OH - C3 - H | H - C4 - OH | H - C5 - OH | H - C6 - O – P | HGlucose 6-phosphate(product)
TRANSFERASES
HexokinaseGlucokinase
OHI
CH-C=C-(CH2)12-CH3H
H0II
R1-C-N-CHI
CH2
H
O
UDP-galact-ose
UDP
Fatty acid
Galactose
Galactosyl transferase
OHI
CH-C=C-(CH2)12-CH3
Ceramide
0II
R1-C-N-CHI
CH2OH
H
H
H
Cerebroside (Galactocerebroside; a glycosphingolipid)
H
O
H OH
HHO
OHH
HOCH2
H
O
TRANSFERASESGlycosyltransferase – if the transferred
group is a carbohydrate residue
HYDROLASESCatalyze cleavage of chemical bonds by
addition of H2O, producing 2 products
O O || ||-O – P ~ O – P ~ O- + HOH | | -O O-
Pyrophosphate (PPi)
O || 2 HO – P – O- | -O Phosphate 2 (Pi)
Pyrophosphatase
Phosphate bond
O O || ||CH3-(CH2)12-C-CH2-C-S-CoA
O || CH3-(CH2)12-C-S-CoA
β-Ketoacyl CoA
Fatty acyl CoA (2 carbons shorter)
Thiolase CH3-C-S-CoA
CoA
Acetyl CoA
HYDROLASESCatalyze cleavage of chemical bonds,
producing 2 products
LYASES
Cleave C-C, C-O, C-N bonds by means other than hydrolysis or oxidation
O O-
\\ / C | C = O H+
| CH3
Pyruvate
H O \ // C + O = C = O | Carbon CH3 dioxide (CO2)
Pyruvatedecarboxylase
Acetaldehyde
LYASES
Dopamine
3,4-Dihydroxyphenylalanine (DOPA)
CH2 – CH – COO-
I NH3
+
OH
HO
CH2 – CH2 – NH3
OH
HO
+
DOPA decarboxylaseCO2
43
PLP
Cleave C-C, C-O, C-N bonds by means other
than hydrolysis or oxidation
H|
CH2 – C – COO-
| NH3
PLP
CH2 – CH2 – NH3
Histidinedecarboxylase
Histidine
HISTAMINE
CO2
LYASESCleave C-C, C-O, C-N bonds by means other
than hydrolysis or oxidation
Catalyze C-C bond cleavage in a reversible reaction
GLYCOLYSIS
P P | | O O H OH OH O | || | | | |H - C1 – C2 – C3 - C4 - C5 - C6 - H | | | | | H OH H H HFructose 1,6-Bisphosphate
O ||H – C1 – H |H – C2 – OH |H – C3 – O – P | H
O ||H – C4 – O – P |H – C5 – OH |H – C6 – OH | H
Glyceraldehyde3 – Phosphate (GADP)
DihydroacetonePhosphate (DHAP)
Aldolase A
Δ G0 = + 5.73 kcal/mole
LYASES
LYASESSynthase – catalyzes a physiologically important reaction that favors the formation of a C-C bond
S~CoA|C1 = O|C2H3
C1OO-
|C2= O|C3H2
|C4OO-
C1OO-
| C2H2
| HO – C3 – C4OO-
| C5H2
| C6OO-
Oxaloacetate (C4)
Acetyl CoA (C2) Citrate (C6)
H2O HS-CoA
Citrate synthase
LYASESSynthase – catalyzes a physiologically important reaction that favors the formation of a C-C bond
HS-CH2-CH2-CH-COO-
I NH3
+
CH2OHI
HC-NH3+
I COO-
Homocysteine
Serine CH2
I CH2
I CH-NH3
+
I COO-
CH2
I H -C-NH3
+
I COO-
S
Cystathione
Cystathionine synthase
H2O
PLP
LYASES
Hydratase – add H2O to a susbtrate
C1OO-
| H – C2
|| C3– H | C4OO-
Fumarate
C1OO-
|HO – C2 – H | H – C3 – H | C4OO-
Malate
Fumarase(or fumarate hydratase
H2O
ISOMERASESTransfer of functional groups or double
bonds within the same molecule
C1OO-
|H3N – C2 – H
| C3H3
L-Alanine
C1OO-
| H – C2 – NH3
| C3H3
D-Alanine
Alanineracemase
Transfer of functional groups or double bonds within the same molecule
GLYCOLYSIS
O ||H – C1 – H |H – C2 – OH |
H – C3 – O – P | H
O ||
H – C1 – O – P |H – C2 – OH |H – C3 – OH | H
Glyceraldehyde3 – Phosphate (GADP)
DihydroacetonePhosphate (DHAP)
ISOMERASES
Triosephosphateisomerase
(Aldose)(Ketose)
O ||
C1 - H
| H - C2 - OH |OH - C3 - H | H - C4 - OH | H - C5 - OH | H - C6 - O – P | H
H | H – C1 - OH |
C2 = O
|OH - C3 - H | H - C4 - OH | H - C5 - OH | H - C6 - O – P | H
Phosphohexoisomerase
Aldehyde group
Keto group
Glucose 6-Phosphate Fructose 6-Phosphate
ATP
ADP
ISOMERASESTransfer of functional groups or double
bonds within the same molecule
O || C1 – O-
|H – C2 – O - P |H – C3 – OH | H
O || C1 – O-
|H – C2 – OH |H – C3 – O – P | H
3-Phosphoglycerate 2-Phosphoglycerate
Phosphoglycerate mutase
Mg+2
Δ G0 = + 1.06 kcal/mole
ISOMERASESTransfer of functional groups or double
bonds within the same molecule
ISOMERASESTransfer of functional groups or double
bonds within the same molecule
C1H2OH I
C2=O I
HOC3H I
HC4OH I
H2C5OPO32-
C1H2OH I
C2=O I
HC3OH I
HC4OH I
H2C5OPO32-
Epimerase
D- Xylulose 5-phosphate D- Ribulose 5-phosphate
C-3 Epimers
CH3
|C = O |COO-
COO-
|CH2
|C = O |COO-Biotin-
CO2
ATPADP + Pi
OxaloacetatePyruvate
Pyruvate carboxylase
ATP ADP+ Pi
LIGASESCatalyze the joining of
substrates in the presence of ATP.
CHARACTERISTICS OF ENZYMES
They are not changed by the reaction they catalyze.
They do not change or alter the equilibrium of the
chemical reaction.
CHARACTERISTICS OF ENZYMES
They increase reaction rates by decreasing
the activation
energy.
CHARACTERISTICS OF ENZYMES
ENZYMES DECREASE THEACTIVATION ENERGY
Reaction progress
ΔGfor the reaction
ΔG
+
++(catalyzed)
ΔG++
(uncatalyzed)
Transition state, S
++
Substratesor
Reactants(e.g. CO2 + H2O)
Products(H2CO3)F
ree
en
erg
y, ∆
GReaction Coordinate Diagram
They are highly specific for the reactants or substrates
they act on and catalyze only one type
of chemical
reaction.
CHARACTERISTICS OF ENZYMES
ENZYME SPECIFICITY
CO2 + H2OH2CO3
Carbonicanhydrase
ENZYME SPECIFICITY
Catalase
2 H2O2 2 H2O
ENZYME SPECIFICITY COO-
| HO - C – H | CH3
COO-
I C – OH
I CH3
Lactate dehydrogenase
L-Lactate PyruvateNAD+ NADH+ + H+
COO-
| H - C – OH | CH3
D-Lactate
ENZYME SPECIFICITY O || C1 - H | H - C2 - OH |OH - C3 - H | H - C4 - OH | H - C5 - OH |
H - C6 – OH
| H
Glucose
O || C1 - H | H - C2 - OH |OH - C3 - H | H - C4 - OH | H - C5 - OH |
H - C6 - O – P
| HGlucose 6-phosphate
Glucokinase
ATP ADP + Pi
Mg+2
CHARACTERISTICS OF ENZYMES
HI
R – C – CO –I
NH2
H I
NH – C – R’ I
COOH
PEPTIDE BOND
They are mostly proteins in nature.
ENZYME-SUBSTRATE COMPLEX FORMATIONThe First Step in Enzymatic Catalysis
Substrate concentration [S]
Rea
ctio
n v
elo
city
(V
)
Maximal velocity
ENZYME-SUBSTRATE COMPLEXCYTOCHROME P450-CAMPHOR
Camphor
Cytochrome P-450
MODELS OF ENZYME-SUBSTRATE COMPLEX
Lock and Key Model (Emil Fischer, 1894) Induced Fit Model (Daniel E. Koshland, Jr, 1958)
LOCK AND KEY MODEL
INDUCED FIT MODEL
KINETICS OF ENZYME-CATALYZED REACTIONS
E + S ES E + Pk1
k-1
k2
k-2
Substrate binding
Catalyticstep
MICHAELIS-MENTEN EQUATION
Vo = Vmax [S]{Km + [S]}
Vo = Velocity at any time (moles/time)Vmax = Maximal velocity (or reaction rate)Km = Michaelis constant for the particular enzyme under investigation = (K-1 + K2)/K1
[S] = Substrate concentration (molar)
MICHAELIS-MENTEN SATURATION CURVE
Substrate concentration [S]
Km
Vmax
2
Vmax
Rea
ctio
n v
elo
city
(V
O)
Zero order
B
A
C
First order
│
│││││││ ││
││
││
││
A = [S] < Km
B = [S] = Vmax/2
C = [S] > Km
= Vo is maximal(Vmax)
A B C
=S
=E
REPRESENTATION OF AN ENZYME IN THE PRESENCE OF A SUBSTRATE
[S] < Km [S] = Vmax/2 [S] > Km
1. It is the substrate concentration at which half of the active sites of the enzyme are filled up.
2. It is an inverse measure of the affinity of the substrate for the enzyme:
a. The lower the Km, the higher is the
affinity.
b. The higher the Km, the lower is the affinity.
SIGNIFICANCE OF KM
VI. Reactions: GLYCOLYSIS
ATP ADP + Pi
Mg+2
O || C1 - H | H - C2 - OH |OH - C3 - H | H - C4 - OH | H - C5 - OH | H - C6 – OH | H
Glucose
O || C1 - H | H - C2 - OH |OH - C3 - H | H - C4 - OH | H - C5 - OH | H - C6 - O – P | HGlucose 6-
phosphate
KM AND PHYSIOLOGICAL UTILIZATION OF GLUCOSE
Hexokinase(Extrahepatic cells, RBCs)
Glucokinase(Liver, Pancreatic β-cells
Km for hexokinase = 0.1 mMKm for glucokinase = 5 mM
LINEWEAVER- BURKE DOUBLE RECIPROCAL PLOT
1
[S]
1V
Intercept on Y-axis = 1
Vmax
Intercept on X-axis =
Slope = Km
Vmax
Km
- 1
LINEWEAVER-BURKE DOUBLE RECIPROCAL PLOT:SAMPLE PROLEM
I I III I II II I I I.050 .25.1 .1
5.2
.3
55
50
45
35
40
30
25
20
15
10
1/[S] (mM-1)
1/V
(m
M s
ec
-1)-
1
Y intercept = 1
Vmax
X intercept = 1
Km
Slope = KmVmax
-
- .12
Y intercept
I IIII
LINEWEAVER-BURKE DOUBLE RECIPROCAL PLOT: SAMPLE PROBLEM
Y intercept = 1
Vmax
Vmax =1
Y intercept
Vmax =1
20
Vmax = 0.05 (mM sec-1)-1
X intercept = -1
Km = -
--
Km
1
.12Km =
8.33 (mM-1)
X intercept
1
Km =
INHIBITION OF ENZYMATIC REACTIONS
Reversible
a. Competitive
b. Non-competitive
c. Uncompetitive
Irreversible
REVERSIBLE INHIBITION
COMPETITIVE INHIBITION: LOVASTATIN
Inhibitor Type Binding Site on Enzyme Kinetic Effect
Competitive Inhibitor
Inhibitor binds specifically at active or catalytic site, where it competes with substrate for binding; inhibition is reversed by increasing substrate concentration.
Vmax unchanged; Km increased to reach a given velocity.
Noncompetitive Inhibitor
Inhibitor binds E or ES other than at the active or catalytic site; substrate binding unaltered but ESI complex cannot form due to structural change in the enzyme →↓ catalytic power no products formed; inhibition cannot be reversed by increasing substrate concentration since inhibitor cannot be driven from the enzyme.
Vmax decreased proportionately to inhibitor concentration; Km unchanged (since substrate can still bind to the enzyme).
Uncompetitive Inhibitor
Inhibitor binds only to ES complexes at locations other than catalytic site; substrate binding modifies enzyme structure, making inhibitor-binding site available; inhibition cannot be reversed by increasing substrate concentration; rare in occurrence.
Vmax decreased
Km decreased
REVERSIBLE INHIBITON
Vmax unchanged by competitive inhibitors since increasing substrate concentration can displace virtually all competitive inhibitors bound to theactive site, hence the identical Y-axis intercepts of Lineweaver-Burke plots, with or without inhibitor.
A competitive inhibitor increases Km for a given substrate in order to attain Vmax that were reached in its absence, hence the differing –X intercepts.
1[S]
1[ V ]
Uninhibited enzyme
Competitiveinhibitor
1Vmax
LINEWEAVER-BURKE PLOT FOR COMPETITIVE INHIBITION
-1Km
Vmax is decreased since a
noncompetitive inhibitor
reduces the concentration of
ES complex that can advance
to reaction products, hence
the differing Y-intercepts.
Km is unchanged since
substrate can still bind to the
enzyme, hence the identical
–X intercepts of Lineweaver-
Burke plots, with or without
noncompetitive inhibitor. 1
[S]
1[ V ]
Uninhibited enzyme
Noncompetitiveinhibitor
LINEWEAVER-BURKE PLOT FOR NONCOMPETITIVE INHIBITION
- 1 Km
1Vmax
Uncompetitive inhibitors
decrease both Vmax and
Km, hence the
production of parallel
lines in uninhibited and
inhibited reactions.
1[S]
1[ V ]
Uninhibited enzyme
Uncompetitiveinhibitor
LINEWEAVER-BURKE PLOT FOR UNCOMPETITIVE INHIBITION
1 Km
-
1Vmax
LINEWEAVER-BURKE DOUBLE RECIROCAL PLOT IN THE PRESENCE OF AN INHIBITOR
1[S]
1[ V ]
1Vmax
Noncompetitive inhibitor
Competitive inhibitor
Uninhibitedenzyme
Uncompetitive inhibitor
-1Km
Type of Inhibition
Vmax
Km
CompetitiveSame ↑
Noncompetitive↓ Same
Uncompetitive↓ ↓
IRREVERSIBLE INHIBITION Diisopropylphosphofluoridate (DIPF)
OII
H3C-C- O-CH2-CH2-N(CH3)3
OH I
Enz-Ser
+ HO- CH2-CH2-N(CH3)3
O I
Enz-Ser
OII
- C – CH3
Acetylcholine Choline
+
OH I
Enz-Ser
H2O
O II
H3C-C-O-
Acetate
A. Normal Reaction of Acetylcholinesterase
A. Reaction with Organophosphorus Inhibitors
OH I
Enz-Ser +
CH3
I H-C-O -
I CH3
CH3
IO-C-O-
I CH3
O II
P - I
F F-, H+
CH3
I H-C-O -
I CH3
CH3
IO-C-O-
I CH3
O II
P - I
O
Enz-Ser
Inactiveenzyme
+
(Enz – acetylcholinesterase)
IRREVERSIBLE INHIBITION - Penicillin
COO-
CH3
N
CS
C
CCH3
H
Penicillin I C=O I H-N I H-C – I C II O
H
I C=O I H-N I H-C – I O=C II
COO-
CH3
N
CS
C
CCH3
H
H
OHI
Ser
Glycopeptidetranspeptidase
OI
Ser
Glycopeptidetranspeptidase
H
Bacterial enzyme
Β-lactam ring
Strained peptide bond
REPRESENTATIVE DRUGS THAT INHIBIT SPECIFIC ENZYMES
DRUG ENZYME TARGET DISEASE
Amrubicin® Topoisomerase II CA chemotherapy
Antabuse® Aldehyde dehydrogenase
Alcoholism
Captopril® Angiotensin-converting enzyme
Hypertension
Celebrex® Cyclooxyenase-2 Arthritis
Digoxin® Na+-K+-ATPase pump Heart problem
Agenerase® HIV protease Acquired Immunodeficiency Syndrome (AIDS)
Lipitor® HMG CoA reductase Hypercholesterolemia
Viagra® Phosphodiesterase Erectile dysfunction
ENZYMES AND PHARMACOTHERAPY: ACE INHIBITORS
Angiotensinogen(liver, polypeptide,
400 AAs)
Renin(JG cells, placenta)
Angiotensin I(decapeptide, 10 AAs)
Angiotensin II(octapeptide, 8 AAs)
Angiotensin converting
enzyme (ACE)
Functions/Effects
Arteriolar vasocons-
triction
Aldosterone-mediated renal
Na+ & H2O reabsorption
↑ BP
ACE Inhibitor (Captopril, Amlodipine)
_
HMG CoA (3-hydroxy-3-methylglutaryl CoA )
2 NADPH + 2H+
2 NADP+
HMG CoA reductase
O OH II | -O – C – CH2 – C – CH2 – CH2OH | CH3
MEVALONATE (C6)
Statins Atorvastatin(Ex. Lipitor)
CoA
O OH O || | || C – CH2 – C – CH2 – C – S-CoA / |O- CH3
ENZYMES AND PHARMACOTHERAPY: STATINS
-
CHOLESTEROL
Viagra® (Sildenafil citrate)
GMP
Smooth muscle relaxation
and vasodilation in penile
blood vessels
SUSTAINED ERECTION
GTP cGMP
Adenylate cyclase
Phospho-diesterase
ENZYMES AND PHARMACOTHERAPY: VIAGRA®
Pi
↑ cGMP
-
ENZYMES AND PHARMACOTHERAPY:COX-2 INHIBITORS
PHOSPHOLIPIDS (Cell Membrane)
Arachidonic acid (C20:Δ4) (Eicosatetraenoic Acid)
5-Lipoxygenase Cyclooxy-genase
PGI2
(Prostacyclin)
TXA2
(ThromboxaneA2)
PGE2
PGF2α
Phospholipase A2
LTC4 LTD4
LTB4
LTE4
LTA4
(Leukotriene A4)
Glu
Glutathione(Glu-Gly-Cys)
ProstaglandinH2 synthase
ProstaglandinH2 synthase
PGG2
(Prostaglandin G2)
Peroxidase
PGH2
(Prostaglandin H2)Prostacyclin
synthaseThromboxane
synthaseIsomerase
Reductase
5-HPETE 5-HETESpontaneousPeroxidase
Leukotriene synthase
Leukotriene synthase
Gly
COX-1 COX-2
-NSAIDS-Selective COX-2 inhibitors (Ex. Celebrex®)
NSAIDS (ASA, Indomethacin)
Pain and inflammation
EICOSANOID SYNTHESIS
--
ENZYMES AND PHARMACOTHERAPY: ANTABUSE®
CH2 – CH2 – OHEthanol
CH3 - OHAcetaldehyde
Alcohol dehydrogenaseNAD+
NADH + H+
Aldehyde dehydrogenase
Acetate
Acetyl CoA
Antabuse®
_
KINETICS FOR AN ALLOSTERIC ENZYME
REGULATION OF ENZYME ACTIVITY
Feedback Inhibition Allosteric (Non-covalent) Modification Covalent Modification Zymogen Activation Induction or Repression of Enzyme Synthesis
FEEDBACK INHIBITIONOriginal Precursor(s)
Enzyme 1
Enzyme 2
Enzyme 3
Enzyme 4
Enzyme 5
1
2
3
4
Final Products
FEEDBACK INHIBITION
Carbamoyl PO4 + Aspartate
Carbamoyl aspartate
Aspartate transcarbamoylase (ATCase)
series of reactions
Cytidine triphosphate (CTP)
RNA & DNA synthesis
FEEDBACK INHIBITION OF HMG CoA REDUCTASE BY CHOLESTEROL
Acetyl CoA
Acetyl CoA
HMG CoA
Mevalonic acid + CoA
HMG CoA reductase
Cholesterol
Feedbackinhibition
Acetoacetyl CoA
2NADPH
+ 2H
+
2NA
DP
++
2H+
> 25 steps
ALLOSTERIC MODIFICATIONAllosteric modulator (activator or inhibitor)
Binds to regulatory or allosteric site
Conformational change in the regulatory enzyme
Effect is transmitted to the active site
Change in shape of the active site
Altered activity
ALLOSTERIC MODIFICATION:Phosphofructokinase I
COVALENT MODIFICATION
ATP ADP
ENZYME- Ser -- OH
HPO4= H2O
Proteinkinase
Phospho-protein
phosphatase
ENZYME- Ser – O – PO32-
COVALENT MODIFICATION OF THE ENZYME
Glycogen phosphorylase
AMPATP
and/orG6P
Glucose
2 ATP 2 ADP
2 H2O2 P
Phosphorylasekinase
Phosphorylase b(inactive)
Phosphorylase a(active)
PP
P P
Phosphoproteinphosphatase
COVALENT MODIFICATION: PYRUVATE DEHYDROGENASE
Pyruvate dehydrogenase
Pyruvatedehydrogenase
P
Pyruvate dehydrogenase
kinase
ATP ADP
Pyruvate dehydrogenase
phosphatasePi H2O
(inactive)(active)
ENZYMES Low activity High activity
Acetyl CoA Carboxylase EP E
Glycogen synthase EP E
Pyruvate dehydrogenase EP E
HMG CoA reductase EP E
Glycogen phosphorylase E EP
Citrate lyase E EP
Phosphorylase b kinase E EP
HMG CoA reductase kinase E EP
MAMMALIAN ENZYMES WHOSE CATALYTIC ACTIVITY IS ALTERED BY COVALENT PHOSPHORYLATION-DEPHOSPHORYLATION
E = Dephosphorylated EP = Phosphoenzyme
ZYMOGEN ACTIVATIONChymotrypsinogen
ZYMOGEN ACTIVATION: BLOOD COAGULATION
Clotting Factors
Prothrombin Thrombin
Fibrinogen Fibrin
Ca+2
Some of the processes involved in blood clotting
INDUCTION OR REPRESSIONOF ENZYME SYNTHESIS
↑ Blood glucoselevels
(Well-fed state)
↑ Insulin
↑ Synthesis of key enzymes involvedin glucose degradation
↓ Blood glucose levels(Starvation)
↑ Glucagon
↑ Synthesis of key enzymes involvedin glucose synthesis
FACTORS AFFECTING ENZYME ACTIVITY
Temperature pH Substrate concentration Co-factors
EFFECT OF TEMPERATURER
eacti
on
velo
cit
y (V
o)
Temperature (oC)
│
││ ││ ││ ││ │ ││ │ ││ ││
││
││
││
││
││
││
70605040302010 80
Optimum T
Heat inactivation
of the enzyme
Increasing enzyme activity
EFFECT OF pH
Optimum pH
OPTIMUM pH OF VARIOUS ENZYMES
EFFECT OF CO-FACTORS: Chlorides, Bromides, Iodides
Cofactors increase the rate of enzyme-catalyzed reactions
EFFECT OF SUBSTRATE CONCENTRATION
Substrate concentration [S]
Vmax
Re
act
ion
ve
loc
ity
(V
)
ENZYMES IN CLINICAL DIAGNOSIS
CARDIAC ENZYMES AS MARKERS FORMYOCARDIAL INFARCTION (MI)
Aspartate aminotransferase
QUESTION 1
Which of the following is TRUE when a substrate concentration equals km in an enzyme-catalyzedreaction? A. A few of the enzyme molecules are present as ES complex.B. Majority of the enzyme molecules are present as ES complex.C. Half of the enzyme molecules are present as ES complex.D. All of the enzyme molecules are present as ES complex.
QUESTION 1
Which of the following is TRUE when a substrate concentration equals km in an enzyme-catalyzedreaction? A. A few of the enzyme molecules are present as ES complex.B. Majority of the enzyme molecules are present as ES complex.C. Half of the enzyme molecules are present as ES complex.D. All of the enzyme molecules are present as ES complex.
Competitive inhibition can be relieved by
increasing which of the following?
A. Enzyme concentration
B. Inhibitor concentration
C. Enzyme-substrate concentration
D. Substrate concentration
QUESTION 2
Competitive inhibition can be relieved by
increasing which of the following?
A. Enzyme concentration
B. Inhibitor concentration
C. Enzyme-substrate concentration
D. Substrate concentration
QUESTION 2
Which of the following enzymes requires
biotin as a coenzyme?
A. PEP carboxykinase
B. Pyruvate carboxylase
C. Phosphofructokinase I
D. Pyruvate dehydrogenase
QUESTION 3
Which of the following enzymes requires
biotin as a coenzyme?
A. PEP carboxykinase
B. Pyruvate carboxylase
C. Phosphofructokinase I
D. Pyruvate dehydrogenase
QUESTION 3
An enzyme with a low Km indicates which of the following? A. High affinity for the substrate.B. Requires increased amount of substrate to become saturated.C. Vmax can be reached at high substrate concentration.D. Less enzyme-substrate complexes are formed.
QUESTION 4
An enzyme with a low Km indicates which of the following? A. High affinity for the substrate.B. Requires increased amount of substrate to become saturated.C. Vmax can be reached at high substrate concentration.D. Less enzyme-substrate complexes are formed.
QUESTION 4
QUESTION 5
Enzymes can be regulated when
phosphorylated. This type of regulation
is called:
A. Allosteric control
B. Feed back regulation
C. Covalent modification
D. Enzyme induction
QUESTION 5
Enzymes can be regulated when
phosphorylated. This type of regulation
is called:
A. Allosteric control
B. Feed back regulation
C. Covalent modification
D. Enzyme induction
REFERENCES
• Lehninger’s Principles of Biochemistry, Nelson, D.L., Cox, M.M., 5th ed., pp. 183-220. • Harper’s Illustrated Biochemistry, Murray, R. K., et. al., 29th ed., pp. 62-82. • Biochemistry with Clinical Correlations, Devlin, M. T., 7th ed., pp. 378-421.• Biochemistry, Lippincott’s Illustrated Reviews, Champe, P.C., Harvey, R.A., 4th ed., pp. 53- 67. • Principles of Biochemistry, Horton, H.R., et al., 4th ed., pp. 129-156. • Marks’ Basic Medical Biochemistry: A Clinical Approach, Lieberman, M., Marks, A.D., 3rd ed., pp. 116-154.
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