Chapter 2 Enzyme
description
Transcript of Chapter 2 Enzyme
Chapter 2
Enzyme
1. Properties of enzymes
2. Structural features of enzymes
3. Mechanism of enzyme-catalyzed reactions
4. Kinetics of enzyme-catalyzed reactions
5. Inhibition of enzymes
6. Regulation of enzymes
7. Clinical applications of enzymes
8. Nomenclature
Contents
Section 1
Properties of Enzymes
A + B → C + D
§ 1.1 General Concepts
GG0RTln[C][D][A][B]
• spontaneous reaction only if G is negative.
• at equilibrium if G is zero.
• spontaneously impossible if G is positive.
Reaction progress
Fre
e e
nerg
y
G forthe reaction
reactants
products
transition state, S
G+ (uncatalyzed)
Catalyzed reactions
• Reactants need to pass over the energy barrier, G+.
• Catalysts reduce the activation energy and assist the reactants to pass over the activation energy.
Reaction progress
Fre
e e
nerg
y
G forthe reaction
reactants
products
transition state, S
G+ (catalyzed)
G+ (uncatalyzed)
1. Fragile structures of the living systems
2. Low kinetic energy of the reactants
3. Low concentration of the reactants
4. Toxicity of catalysts
5. Complexity of the biological systems
Chemical reactions in living systems are quite different from that in the industrial situations because of
Need for special catalysts
• Enzymes are catalysts that have special characteristics to facilitate the biochemical reactions in the biological systems.
• Enzyme-catalyzed reactions take place usually under relatively mild conditions.
Enzymes
§ 1.2 Characteristics
Enzyme-catalyzed reactions have the following characteristics in comparison with the general catalyzed reactions:
• common features: 2 “do” and 2 “don’t”
• unique features: 3 “high”
• Do not consume themselves: no changes in quantity and quality before and after the reactions.
• Do not change the equilibrium points: only enhance the reaction rates.
• Apply to the thermodynamically allowable reactions
• Reduce the activation energy
Common features
• Enzyme-catalyzed reactions have very high catalytic efficiency.
• Enzymes have a high degree of specificity for their substrates.
• Enzymatic activities are highly regulated in response to the external changes.
Unique features
CatalystActivation energy
(cal/M)
No catalyst 18,000
Normal catalyst 11,700
Hydrogen peroxidase 2,000
§ 1.3.a High efficiency
Accelerated reaction rates
enzyme
Non-enzymatic
rate constant
(kn in s-1)
enzymatic
rate constant
(kn in s-1)
accelerated reaction rate
Carbonic anhydrase 10-1 106 8 x 106
Chymotrypsin 4 x 10-9 4 x 10-2 10-2107
Lysozyme 3 x 10-9 5 x 10-1 2 x 108
Triose phosphate isomerase
4 x 10-6 4 x 103 109
Urease 3 x 10-10 3 x 104 1014
Mandelate racemase 3 x 10-10 5 x 102 1.7 x 1015
Alkaline phosphatase
10-15 102 1017
• Absolute specificity
• Relative specificity
• Stereospecificity
§ 1.3.b High specificity
Unlike conventional catalysts, enzymes demonstrate the ability to distinguish different substrates. There are three types of substrate specificities.
Absolute specificity
Enzymes can recognize only one type of substrate and implement their catalytic functions.
O C
NH2
NH2
+ H2O 2NH3 + CO2
urea
urease
O C
NH
NH2
+ H2O
methyl urea
CH3
Enzymes catalyze one class of substrates or one kind of chemical bond in the same type.
Relative specificity
protein kinase Aprotein kinase Cprotein kinase G
To phopharylate the -OH group of serine and threonine in the substrate proteins, leading to the activation of proteins.
OH
OH
HH
OHH
OH
CH2OH
H
CH2OH
HCH2OH
OH H
H OH
O
O
1
1
OH
OH
HH
OHH
OH
CH2
H
CH2OH
HCH2OH
OH H
H OH
O
O
1
1
O
OOH
H
HH
OHH
OH
CH2OH
H 1
sucrose
raffinose
sucrase
StereospecificityThe enzyme can act on only one form of isomers of the substrates.
H
C
H3C COOHOH
H
C
H3C OHCOOH
AB C A
B C
Lactate dehydrogenase can recognize only the L-form but the D-form lactate.
• Enzyme-catalyzed reactions can be regulated in response to the external stimuli, satisfying the needs of biological processes.
• Regulations can be accomplished through varying the enzyme quantity, adjusting the enzymatic activity, or changing the substrate concentration.
§ 1.3.c High regulation
Section 2
Components of Enzymes
• Almost all the enzymes are proteins having well defined structures.
• Some functional groups are close enough in space to form a portion called the active center.
• Active centers look like a cleft or a crevice.
• Active centers are hydrophobic.
§ 2.1 Active Center
Lysozyme
Residues (colored ) in the active site come from different parts of the polypeptide chain .
• Binding group: to associate with the reactants to form an enzyme-substrate complex
• Catalytic group: to catalyze the reactions and convert substrates into products
Two essential groups
The active center has two essential groups in general.
+- Catalytic group
Binding group
Substratemolecule
Protein chain
Active center
Essential groupsoutside theactive center
Active centers
• Simple enzymes: consists of only one peptide chain
• Conjugated enzymes:
holoenzyme = apoenzyme + cofactor
(protein) (non-protein)
• Cofactors: metal ions; small organic molecules
§ 2.2 Molecular Components
Metal ions
• Metal-activated enzyme: ions necessary but loosely bound. Often found in metal-activated enzyme.
• Metalloenzymes: Ions tightly bound.
• Particularly in the active center, transfer electrons, bridge the enzyme and substrates, stabilize enzyme conformation, neutralize the anions.
• Small size and chemically stable compounds
• Transferring electrons, protons and other groups
• Vitamin-like or vitamin-containing molecule
Organic compounds
• Loosely bind to apoenzyme. Be able to be separated with dialysis.
• Accepting H+ or group and leaving to transfer it to others, or vise versa.
Coenzymes
Prosthetic groups• Tightly bind through either covalent or many n
on-covalent interactions.
• Remained bound to the apoenzyme during the course of reaction.
Section 3
Mechanism of Enzyme-Catalyzed Reactions
• Proximity and orientation arrangement
• Multielement catalysis
• Surface effect
To understand the molecular details of the catalyzed reaction.
Lock-and-key model
Both E and S are rigid and fixed, so they must be complementary to each other perfectly in order to have a right match.
Induced-fit model
The binding induces conformational changes of both E and S, forcing them to get a perfect match.
Hexokinase catalyzing glycolysis
• Hexokinase, the first enzyme in the glycolysis pathway, converted glucose to glucose-6-phosphate with consuming one ATP molecule.
• Two structural domains are connected by a hinge.
• Upon binding of a glucose molecule, domains close, shielding the active site for water.
Induced structural changes
Section 4
Kinetics of Enzyme- Catalyzed Reactions
§ 4.1 Reaction rate
Time (t)0
[P]
[P]
t
Initial slope = vo =[P]
t
• The reaction rate is defined as the product formation per unit time.
• The slope of product concentration ([P]) against the time in a graphic representation is called initial velocity.
• It is of rectangular hyperbolic shape.
Initial velocity
Reaction velocity curve
[S]0
Vmax
V0
Vmax/2
Km
Intermediate state
Forming an enzyme-substrate complex, a transition state, is a key step in the catalytic reaction.
initial intermediate final
k3k1
k2
E S E + PE + S
Reaction progress
Fre
e e
nerg
y
G forthe reaction
reactants
products
transition state, S
G+ (catalyzed)
G+ (uncatalyzed)
• K1 = rate constant for ES formation
• K2 = rate constant for ES dissociation
• K3 = rate constant for the product released from the active site
Rate constants
k3k1
k2
E S E + PE + S
• The mathematical expression of the product formation with respect to the experimental parameters
• Michaelis-Menten equation describes the relationship between the reaction rate and substrate concentration [S].
§ 4.2 Michaelis-Menten Equation
• [S] >> [E], changes of [S] is negligible.
• K2 is negligible compared with K1.
• Steady-state: the rate of E-S complex formation is equal to the rate of its disassociation (backward E + S and forward to E + P)
Assumptions
Describing a hyperbolic curve.
Km is a characteristic constant of E
[S] << Km 时, v [S]∝ [S] >> Km 时, v ≈ Vmax
Vmax=V Km + [S]
[S]
[S]0
Vmax
V0
First order withrespect to [S]
Zero order withrespect to [S]
• the substrate concentration at which enzyme-catalyzed reaction proceeds at one-half of its maximum velocity
• Km is independent of [E]. It is determined by the structure of E, the substrate and environmental conditions (pH, T, ionic strength, …)
Significance of Km
[S]0
Vmax
V0
Vmax/2
Km
• Km is a characteristic constant of E.
• The value of Km quantifies the affinity of the enzyme and the substrate under the condition of K3 << K2. The larger the Km, the smaller the affinity.
=Km k1
k3k2 +
Km for selected enzymes
Enzyme Substrate km
Catalase H2O225
Hexokinase ATP 0.4
D-Glucose 0.05
D-Fructose 1.5
Carbonic anhydrase HCO3- 9
Chemotrypsin Glycyltyrosinylglycine 108
N-Benzoyltyrosinamide 2.5
Galactosidase D-Lactose 4
Threonine dehydratase L-Threonine 5
• The reaction velocity of an enzymatic reaction when the binding sites of E are saturated with substrates.
• It is proportional to [E].
Significance of Vmax
• Vmax is the reaction rate when the enzymes are saturated, and is independent of the enzyme concentration.
• The number of the products converted in a unit time by one enzyme molecule which is saturated.
Turnover number
k3 = Vmax / [E]
• To determine Km and Vmax
• To identify the reversible repression
Lineweaver-Burk plot
1=
Km 1
[S]+
Vmax
1V Vmax
Slope = Km/Vmax
1/[S]
1/V
Intercept = 1/Vmax
Intercept = -1/ Km
Double-reciprocal plot
• Substrate concentration
• Enzyme concentration
• Temperature
• pH
• Inhibitors
• Activators
§ 4.3 Factors affecting enzyme-catalyzed reaction
§ 4.3.a Effect of substrate
• Has been described already.
• [E] affects the rate of enzyme-catalyzed reactions
• [S] is held constant.
• When [S] >> [E], V ≈ [E]
§ 4.3.b Effect of enzyme
Re
actio
n v
elo
city
Enzyme concentration
§ 4.3.c Effect of temperature
• Optimal temperature (To) is the characteristic T at which an enzyme has the maximal catalytic power.
• 35 ~ 40C for warm blood species.
• Reaction rates increase by 2 folds for every 10C rise.
• Higher T will denature the enzyme.
Temp. (C)
Enz
ymat
ic a
ctiv
ity
0.5
1.0
2.0
1.5
10 6050403020
§ 4.3.d Effect of pH
• Optimal pH is the characteristic pH at which the enzyme has the maximal catalytic power.
• pH7.0 is suitable for most enzymes.
• Particular examples: pH (pepsin) = 1.8 pH (trypsin) = 7.8
En
zym
atic
act
ivity
1.0
2.0
1.5
0.5
2.0 10.08.04.0 6.0 pH
pepsin
trypsin
Section 5
Inhibition of Enzyme
• Inhibitors are certain molecules that can decrease the catalytic rate of an enzyme-catalyzed reaction.
• Inhibitors can be normal body metabolites and foreign substances (drugs and toxins).
§ 5.1 Inhibitors
• The inhibition process can be either irreversible or reversible.
• The inhibition can be competitive, non-competitive, or un-competitive.
Inhibition processes
• Inhibitors are covalently bound to the essential groups of enzymes.
• Inhibitors cannot be removed with Inhibitors cannot be removed with simple dialysis or super-filtration. simple dialysis or super-filtration.
• Binding can cause a partial loss or complete loss of the enzymatic activity.
§ 5.2 Irreversible inhibition
Acetylcholine accumulation will cause excitement of the parasympathetic system: omitting, sweating, muscle trembling, pupil contraction
Pesticide poisoning
acetylcholine choline + acetic acid
choline esterase
+ E OHRO
PO
X
R'O
RO
PO
O
R'O
E
organophosphate inhibited AChE acid
E OH
OR
P
O OR'
N
CH3
CHNOH+
N
CH3
CHNO+
PAM
AChE
HX+
• Heavy metal containing chemicals bind to the –SH groups to inactivate the enzymes.
Heavy metal poisoning
E
SH
SH
E
S
S
Hg
E
SH
SH
ClAs
Cl
CH
CHCl As CH
CHClE
S
S
Hg2++ + 2H+
+ 2HCl+
• Inhibitors are bound to enzymes non-covalently.
• The reversible inhibition is characterized by an equilibrium between free enzymes and inhibitor-bound enzymes.
§ 5.3 Reversible inhibition
+
S P
I
ES E+
+
EI
E
§ 5.3.a Competitive inhibition
• Competitive inhibitors share the structural similarities with that of substrates.
• Competitive inhibitors compete for the active sites with the normal substrates.
• Inhibition depends on the affinity of enzymes and the ratio of [E] to [S].
V =Vmax [S]
Km(1 + + [S]Ki
[ I ])
Vmax
1=
Km 1
[S] +
Vmax
1 (1 + )
V
[ I ]
Ki
1/[S]
1/V
No inhibitor
Competitiveinhibitorincrease
-1/ Km
-1/ Km(1 + [I]/Ki)
1/Vmax
Lineweaver-Burk plot
• As [S] increases, the effect of inhibitors is reduced, leading to no change in Vmax.
• Due to the competition for the binding sites, Km rises, equivalent to the reduction of the affinity.
Inhibition features
• FH4 (tetrahydrofolate) is a coenzyme in the nucleic acid synthesis, and FH2 (dihydrofolate) is the precursor of FH4.
• Bacteria cannot absorb folic acid directly from environment.
• Bacteria use p-amino-benzoic acid (PABA), Glu and dihydropterin to synthesize FH2.
• Sulfanilamide derivatives share the structural similarity with PABA, blocking the FH2 formation as a competitive inhibitor.
Example-1: competitive inhibitor
Glu
H2N COOH
dihydropterin
FH2 synthetase
FH2 FH4
H2N SO2NHR
Sulfanilamide
Methotrexate
PABA FH2reductase
+
+
COOH
H2C
COOH
malonic acid
HC
COOH
CH
HOOC
succinate
succinate dehydrogenase
fumaric acid
CO-COOH
H2C
COOH
oxaloacetate
H2C
COOH
CH2
HOOC
Example-2: competitive inhibitor
+S P
I
+
+
E
EI
I
+
S+
E
ESI
ES
§ 5.3.b Non-competitive inhibition
• Inhibitors bind to other sites rather than the active sites on the free enzymes or the E-S complexes.
• The E-I complex formation does not affect the binding of substrates.
• The E-I-S complexes do not proceed to form products.
• Reducing the [E-S]
• Vmax↓; unchanged Km.
1/[S]
1/V
No inhibitor
noncompetitiveinhibitorincrease
-1/ Km
(1 + [I]/Ki)/Vmax
Vmax
1=
Km 1
[S] +
Vmax
1 (1 + )
V
[ I ]
Ki (1 + )
[ I ]
Ki
+S P
+E
I
+
E
ESI
ES
§ 5.3.c Uncompetitive inhibition
• Uncompetitive inhibitors bind only to the enzyme-substrate complexes.
• The E-I-S complexes do not proceed to form products.
• The E-I-S complexes do not backward to the substrates and enzymes.
• This inhibition has the effects on reducing both Vmax and Km.
• Commonly in the multiple substrate reactions.
1/[S]
1/V
No inhibitor
Uncompetitiveinhibitorincrease
-1/ Km
(1 + [I]/Ki)/Vmax
1/Vmax
-1/ Km(1 + [I]/Ki)
Vmax
1=
Km 1
[S] +
Vmax
1
V (1 + )
[ I ]
Ki
type binding target Km Vmax
Competitive E only =
Noncompetitive E or ES =
Uncompetitive ES only
Summary of inhibition
Activators are the compounds which bind to an enzyme or an enzyme-substrate complex to enhance the enzymatic activity without being modified by the enzymes.
Activator
• Metal ions
• essential activators: no enzymatic activity without it
Mg2+ of hexokinase
• non-essential activators: enhancing the catalytic power.
Activators
• Enzymatic activity is a measure of the capability of an enzyme of catalyzing a chemical reaction.
• It directly affects the reaction rate.
• International unit (IU): the amount of enzyme required to convert 1 µmol of substrate to product per minute under a designated condition.
Enzymatic activity
• Determination of the enzymatic activity requires proper treatment of enzymes, excess amount of substrate, optimal T and pH, …
• One katal is the amount of enzyme that converts 1 mol of substrate per second.
• IU = 16.67×10-9 kat
• In addition to enzymes, other chemical species often participate in the catalysis.
• Cofactor: chemical species required by inactive apoenzymes (protein only) to convert themselves to active holoenzymes.
§ 2.2 Molecular Components
Cofactors
Activator ions(loosely bound)
Metal ions ofmetalloenzymes(tightly bound)
Cosubstrates(loosely bound)
Prostheticgroups
(tightly bound)
Essential ions Coenzymes
Cofactors
• Activator ions: loosely and reversibly bound, often participate in the binding of substrates.
• Metal ions of metalloenzymes: tightly bound, and frequently participate directly in catalytic reactions.
Essential ions
• Transfer electron
• Linkage of S and E;• Keep conformation of E-S complex
• Neutralize anion
Function of metal ions
• Act as group-transfer reagents to supply active sites with reactive groups not present on the side chains of amino acids
• Cosubstrates:
• Prosthetic groups:
Coenzymes
• The substrates in nature.
• Their structures are altered for subsequent reactions.
• Shuttle mobile metabolic groups among different enzyme-catalyzed reactions.
Cosubstrates
• Supply the active sites with reactive groups not present on the side chains of AA residues.
• Can be either covalently attached to its apoenzymes or through many non-covalent interactions.
• Remained bound to the enzyme during the course of the reaction.
Prosthetic groups
• Metabolite coenzymes: they are synthesized from the common metabolites. – several NTP, ATP (most abundant), UDP-
glucose
• Vitamin-derived coenzymes: they are derivatives of vitamins, and can only be obtained from nutrients. – NAD and NADP+, FAD and FMN, lipid
vitamins, …
Coenzymes
• Until recently, all the enzymes are known to be proteins.
• Ribonucleic acids also demonstrate the catalytic ability.
• Ribozymes have the ability to self-cleave.
• They are highly conservative, an indication of the biological evolution and the primary enzyme.
§ 2.3 Ribozyme
Family of serine protease
Hydrolysissite
Trypsin ElastaseChymotrypsin
Hydrolysissite
Hydrolysissite
§ 5.3.a Competitive inhibition
E + S E + P+I
EI
ES
Ki
§ 5.3.b Non-competitive inhibition
E + S E + P+I
EI + S
ES
Ki
+I
EIS
Ki
§ 5.3.c Uncompetitive inhibition
E + S E + PES+I
EIS
Ki
Section 6
Regulation of Enzyme
• Many biological processes take place at a specific time; at a specific location and at a specific speed.
• The catalytic capacity is the product of the enzyme concentration and their intrinsic catalytic efficiency.
• The key step of this process is to regulate either the enzymatic activity or the enzyme quantity.
• Maintenance of an ordered state in a timely fashion and without wasting resources
• Conservation of energy to consume just enough nutrients
• Rapid adjustment in response to environmental changes
Reasons for regulation
Controlling an enzyme that catalyzes the rate-limiting reaction will regulate the entire metabolic pathway, making the biosystem control more efficient.
Rate limiting reaction is the reaction whose rate set by an enzyme will dictate the whole pathway, namely, the slowest one or the “bottleneck” step.
• Zymogen activation
• Allosteric regulation
• Covalent modification
§6.1 Regulation of E Activity
• Certain proteins are synthesized and secreted as an inactive precursor of an enzyme, called zymogen.
• Selective proteolysis of these precursors leads to conformational changes, and activates these enzymes.
• It is the conformational changes that either form an active site of the enzyme or expose the active site to the substrates.
§6.1.a Zymogen activation
• Hormones: proinsulin
• Digestive proteins: trypsinogen, …
• Funtional proteins: factors of blood clotting and clot dissolution
• Connective tissue proteins: procollagen
Wide varieties
Activation of chymotrypsin
1
1
S
14913 24514614 15 16
149 24514613 14 15 16
1 14616 149 24513
S S S
Pro-CT
CT
CT
• A cascade reaction in general
• To protect the zymogens from being digested
• To exert function in appropriate time and location
• Store and transport enzymes
Features of zymogen activation
• Allosteric enzymes are those whose activity can be adjusted by reversible, non-covalent binding of a specific modulator to the regulatory sites, specific sites on the surface of enzymes.
• Allosteric enzymes are normally composed of multiple subunits which can be either identical or different.
§6.1.b Allosteric regulation
• The multiple subunits are catalytic subunits regulatory subunits
• Kinetic plot of v versus [S] is sigmoidal shape.
• Demonstrating either positive or negative cooperative effect.
• There are two conformational forms, T and R, which are in equilibrium.
• Modulators and substrates can bind to the R form only; the inhibitors can bind to the T form.
Properties of allosteric enzymes
[S]
Allosteric enzyme
Allosteric represion
Allosteric activation
Allosteric curve
Activation of protein kinase
C: catalytic portions
R: regulatory portions
4 cAMP
protein kinase(inactive)
protein kinase(active)
+ +C
C
R
R
C
C
R
R
cAMP
cAMP
cAMP
cAMP
• A variety of chemical groups on enzymes could be modified in a reversible and covalent manner.
• Such modification can lead to the changes of the enzymatic activity.
§6.1.c Covalent modification
phosphorylation - dephosphorylation
adenylation - deadenylation
methylation - demethylation
uridylation - deuridylation
ribosylation - deribosylation
acetylation - deacetylation
Common modifications
Phosphorylation
E-OH E-O-PO3H2
ATP ADP
proteinkinase
phosphorylation
dephosphorylation
H2OPi
Mg2+
phosphatase
• Two active forms (high and low)
• Covalent modification
• Energy needed
• Amplification cascade
• Some enzymes can be controlled by allosteric and covalent modification.
Features of covalent modification
• Constitutive enzymes (house-keeping): enzymes whose concentration essentially remains constant over time
• Adaptive enzymes: enzymes whose quantity fluctuate as body needs and well-regulated.
• Regulation of enzyme quantity is accomplished through the control of the genes expression.
§6.2 Regulation of E Quantity
• Inducer: substrates or structurally related compounds that can initiate the enzyme synthesis
• Repressor: compounds that can curtail the synthesis of enzymes in an anabolic pathway in response to the excess of an metabolite
• Both are cis elements, trans-acting regulatory proteins, and specific DNA sequences located upstream of genes
Controlling the synthesis
• Enzymes are immortal, and have a wide range of lifetime. LDH4 5-6 days, amylase 3-5 hours.
• They degrade once not needed through proteolytic degradation.
• The degradation speed can be influenced by the presence of ligands such as substrates, coenzymes, and metal ions, nutrients and hormones.
Controlling the degradation
• Lysosomic pathway: – Under the acidic condition in lysosomes– No ATP required– Indiscriminative digestion – Digesting the invading or long lifetime proteins
• Non-lysosomic pathway:– Digest the proteins of short lifetime – Labeling by ubiquitin followed by hydrolysis– ATP needed
Degradation pathway
Enzymes/pathways in cellular organelles
organelle Enzyme/metabolic pathway
Cytoplasm Aminotransferases, peptidases, glycolysis, hexose monophosphate shunt, fatty acids synthesis, purine and pyrimidine catabolism
Mitochondria Fatty acid oxidation, amino acid oxidation, Krebs cycle, urea synthesis, electron transport chain and oxidative phosphorylation
Nucleus Biosynthesis of DNA and RNA
Endoplasmic reticulum
Protein biosynthesis, triacylglycerol and phospholipids synthesis, steroid synthesis and reduction, cytochrome P450, esterase
Lysosomes Lysozyme, phosphatases, phospholipases, proteases, lipases, nucleases
Golgi apparatus Glucose 6-phosphatase, 5’-nucleotidase, glucosyl- and galactosyl-transferase
Peroxisomes Calatase, urate oxidase, D-amino acid oxidase, long chain fatty acid oxidase
Section 7
Clinical Applications
• Plasma specific or plasma functional enzymes: Normally present in the plasma and have specific functions.
• High activities in plasma than in the tissues. Synthesized in liver and enter the circulation.
• Impairment in liver function or genetic disorder leads to a fall in the activities.
§7.1 Fundamental Concepts
• Non-plasma specific or plasma non-functional enzymes: either totally absent or at a low concentration in plasma
• In the normal turnover of cells, intracellular enzymes are released into blood stream.
• An organ damaged by diseases may elevate those enzymes
• A group of enzymes that catalyze the same reaction but differ from each other in their structure, substrate affinity, Vmax, and regulatory properties.
• Due to gene differentiation: the different gene products or different peptides of the same gene
• Present in different tissues of the same system, or subcellular components of the same cell
§7.2 Isoenzyme
• Synthesized from different genes (malate dehydrogenase in cytosol versus in mitochondria)
• Oligomeric forms of more than one type of subunits (lactate dehydrogenase)
• Different carbohydrate content (alkaline phosphatase)
Reasons for isoenzyme
• 5 isoenzymes, LDH1 – LDH5
• Tetramer– M subunits (M for muscle), basic – H subunits (H for heart), acidic
• Different catalytic activities
• Used as the marker for disease diagnosis
Lactate dehydrogenase (LDH)
H4
LDH1
M subunitH subunit
LDH2 LDH3 LDH4 LDH5
H1M3 M4H2M2H3M1
• LDH1 (H4) in heart muscle converts lactate to pyruvate, and then to acetyl CoA.
• LDH5 (M4) in skeletal muscle converts pyruvate to lactate.
H3CHC COOH
OH
H3C C COOH
O
LDH
NAD+ NADH + HLactate Pyruvate
• 3 isoenzymes, BB, BM, MM
• Dimeric form: M (muscle) or B (brain)
• CPK2 is undetectable (<2%) in serum for healthy individuals, and elevated to 20% in the first 6-18 hrs after myocardial infarction.
• Used as a earliest reliable indicator of myocardial infarction.
Creatine phosphokinase
• Usefulness:– Enzyme assays provide important information
concerning the presence and severity of diseases
– Provide a means of monitoring the patient’s response
• approaches:– Measuring the enzymatic activities directly
– Used as agents to monitor the presence of substrates
§7.3 Diagnostic Applications
Enzymatic activity changes
CK
act
ivity
1 2 3 4
Rat
io o
f LD
H1 to
LD
H2
LDH1 / LDH2
CM2
CM3
Days after myocardial infarction
1.00
1.25
0.75
Electrophoresis of LDH
Serum enzymes (elevated)
Diseases
Amylase Acute pancreatitis
Serum glutamate pyruvate transaminase (SGPT)
Liver diseases (hepatitis)
Serum glutamate oxaloacetate transaminase (SGOT)
Heart attack (myocardial infarction)
Alkaline phosphatase Rickets, obstructive jaundice
Acid phosphatase Cancer of prostate gland
Lactate dehydrogenase (LDH) Heart attack, liver diseases
γ-glutamyl transpeptidase (GGT) Alcoholism
5’-nucleotidase Hepatitis
Aldolase Muscular dystrophy
Enzymes for disease diagnosis
• Successful therapeutic uses– Steptokinase: treating myocardial infarction; preventing
the heart damage once administrated immediately after heart attack
– Asparaginase: tumor regression
• Several limits– Can be rapidly inactivated or digested
– May provoke allergic effects
§7.4 Therapeutic Applications
Section 8
Nomenclature
• Adding the suffix –ase to the name of the substrates (urease)
• Adding the suffix –ase to a descriptive term for the reactions they catalyze (glutemate dehydrogenase)
• For historic names (trypsin, amylase)
• Being named after their genes (Rec A –recA, HSP70)
§8.1 Conventional Nomenclature
• The International Union of Biochemistry and Molecular Biology (IUBMB) maintains the classification scheme.
• Categorize in to 6 classes according to the general class of organic reactions catalyzed
• Assigned a unique number, a systematic name, a shorter common name to each enzyme
§8.2 Systematic Nomenclature
1. Catalyzing a variety of oxidation-reduction reactions
AH2 + B → A + BH2
2. Alcohol dehydrogenase (alcohol:NAD+ oxidoreductase, E.C. 1.1.1.1.)
Cytochrome oxidase
L- and D-amino acid oxidase
§8.2.a Oxidoreductases
1. Catalyzing transfer of a groups between donors and acceptors
A-X + B → A + B-X
2. Hexokinase (ATP:D-hexose 6-phosphotransferase, E.C.2.7.1.1.)
Transaminase
Transmethylases
§8.2.b Transferases
1. Catalyzing cleavage of bonds by addition of water
A-B + H2O → AH + BOH
2. Lipase (triacylglycerol acyl hydrolase, E.C. 3.1.1.3.)Choline esteraseAcid and alkaline phosphatasesUrease
§8.2.c Hydrolases
1. Catalyzing lysis of a substrate and generating a double bond (nonhydrolytic, and non-oxidative reactions)
A-B + X-Y → AX + BY
2. Aldolase (ketose 1-phosphate aldehyde lysase, E.C. 4.1.2.7.)FumaraseHistidase
§8.2.d Lysases
1. Catalyzing recemization of optical or geometric isomers
A → A’
2. Triose phosphate isomerase (D-glyceraaldehyde 3-phosphate ketoisomerase, E.C. 5.3.1.1.)
Retinol isomerasePhosphohexose isomerase
§8.2.e Isomerases
1. Catalyzing synthetic reactions at the expense of a high energy bond of ATP
A + B → A-B
2. Glutamine synthetase (L-glutamate ammonia ligase, E.C. 6.3.1.2.)
Acetyl CoA carboxylase
Auccinate thiokinase
§8.2.f Ligases
• Blood clot formation and tissue repair are brought “on-line” only in response to pressing physiological or pathophysiological needs.