Enzymes
Enzymes
Enzymes
Enzymes
are biological catalysts that speed up the rate of chemical
reactions
provide a fantastic speed, specificity, and regulatory control to
reactions in the body
Chemical Nature of Enzymes (Properties)
Enzymes are proteins (except ribozymes)
reusable(not consumed or permanently changed) by the reaction
act repeatedly to increase the rate of reactions
are often very “specific” – promote only 1 particular reaction
acts up on reactants called substrates
They have enormous power of catalysis. They increase rate of
reaction to 105 to 1010 folds.
Enzymes do not alter the equilibrium constant (Keq). 1
How enzymes work?
For a chemical reaction A → B to occur, energy is required.
When enough energy is supplied, A undergoes to transition
state which is an unstable state. So, it gets converted to product
B which is more stable.
The amount of energy needed to convert a substance from
ground state to transition state is called activation energy
Enzymes accelerates the rate of reaction by decreasing the
energy of activation
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Enzyme Specificity
Enzymes have varying degrees of specificity for substrates
Enzymes may recognize and catalyze:
- a single substrate
- a group of similar substrates
- a particular type of bond
- a particular site on a substrate
- a particular stereoisomer
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Enzyme Specificity examples….continue
Substrate Specificity
Enzymes are specific towards their substrates
Example,
Reaction Specificity
A given enzyme catalyze only one specific reaction. example,
lipases only hydrolyze lipids, Proteases hydrolyzes proteins, do
not catalyze any other types of reactions.
Optical Specificity/ Sterospecificity
enzymes are able to recognize optical isomers of the substrate.
For example, enzymes of amino acid metabolism act only on L-
isomers (L-amino acid) but not D-isomers (D-amino acids). 4
Group/ broad Specificity
Some lytic (hydrolases) enzymes act on specific groups. e.g.
Proteases are specific for peptide groups, glycosidases are
specific to glycosidic bonds.
Absolute Group Specificity
Certain lytic enzymes exhibit high order group specificity. For
example, chymotrypsin is protein splitting enzyme i.e., it
hydrolyzes peptide bonds. But it preferentially hydrolyzes
peptide bonds in which carboxyl group is contributed by
aromatic amino acids phenylalanine, tyrosine and tryptophan.
Likewise, trypsin another peptide bond hydrolyzing enzyme
preferentially hydrolyzes peptide bonds in which carboxyl
group is contributed by basic amino acids.
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Enzyme Classification and Nomenclature
1. Recommended names
Enzymes were assigned various trivial names as they were
discovered, which are convenient to use.
The name of an enzyme has two parts.
The first part indicates name of its substrate and second part
ending in ‘ase’
For example; Sucrase, Protease, Lipase
2. Systematic Name
International Union of Biochemistry classified all enzymes
into six major classes based on the type of reaction they
catalyze and reaction mechanism
Each enzyme has code (EC) number.
EC is a four-digit number. The first digit indicates major
class, second digit indicates sub class, third digit denotes 6
sub-sub class and final digit indicates specific enzyme.
Example, the formal systematic name of the enzyme catalyzing
the reaction;
ATP + D-glucose → ADP + D-glucose 6-phosphate is: “ATP: D-
hexose-6-phospho-transferase”, which indicates that it catalyzes
the transfer of a phosphoryl group from ATP to glucose.
• Its Enzyme Commission number (E.C. number) is 2.7.1.1. Here,
a member of class 2 (transferase)
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Enzyme Cofactors
Cofactors are non-protein molecules required for activity of
some enzymes
are two types of cofactors:
Organic cofactors(co-enzymes) such as NAD+, FAD+, CoA
Inorganic cofactors(metal Ions) such as Fe 2+, Mg2+, Mn2+, or
Zn2+
A coenzyme or metal ion that is very tightly or even covalently
bound to the enzyme protein is called a prosthetic group.
A complete, catalytically active enzyme together with its bound
coenzyme and/or metal ions is called a holoenzyme.
The protein part of such an enzyme is called the apoenzyme or
apoprotein.
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Isoenzymes (Isozymes)
These are enzymes that catalyze the same reaction but
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Enzyme Kinetics
enzyme concentration
Temperature
Inhibitors
Activators(cofactors)
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1. Effect of substrate
The rate of an enzyme-catalyzed reaction increases with
substrate concentration until a maximal velocity (Vmax) is reached
At low substrate concentrations a doubling of [S] will lead to a
doubling of the initial velocity (V0).
At higher substrate concentrations the enzyme becomes saturated
and further increases in [S] lead to very small changes in V0.
This occurs because, at saturating substrate concentrations
effectively all of the enzyme molecules have bound substrate. The
overall enzyme rate is now dependent on the rate at which the
product can dissociate from the enzyme,
The shape of the resulting graph when V0 is plotted against [S] is
called a hyperbolic curve
The simplest of these equations, the Michaelis-Menten equation,
relates the initial velocity (V0) to the concentration of substrate
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3. Temperature
Temperature affects the rate of reactions in two ways.
• First, arise in temperature increases the thermal energy of the
substrate molecules. This raises the proportion of substrate
molecules with sufficient energy to overcome energy of
activation, and hence increases the rate of the reaction.
A second effect comes into play at higher temperatures.
Increasing the thermal energy of the molecules increase the
chances of breaking the multiple weak, non-covalent interactions
Ultimately this will lead to the denaturation (unfolding) of the
enzyme (human E start at >40oc)., decreasing enzyme activity
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4. pH
Each enzyme has an optimum pH
Slight deviations in the pH from the optimum lead to a decrease
in the reaction rate.
Larger deviations in pH lead to denaturation of the enzyme
due to changes in the ionization of amino acid residues and
the disruption of non covalent interactions.
Many enzymes have a pH optimum of around 6-8,
but there is great diversity in the pH optima of enzymes, due to
the different environments.
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Enzyme inhibition
5. Inhibitors
Any substance that can diminish the velocity of an enzyme-
catalyzed reaction is called an inhibitor
Enzyme inhibition can be of two main types:
irreversible or
reversible.
Uncompetitive inhibition
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1. Irreversible Inhibitors
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2. Reversible Inhibitors
Inhibitor bind to enzyme by non-covalent interaction and are
subsequently released
a. Competitive inhibitors
Shape and structure of inhibitor is very similar to substrate
Inhibitor mimic substrate (or transition state) and fits into the
active site. Physically blocks substrate’s access into the active site
Effects of competition can be overcome by increasing [S].
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Example 1. Allopurinol
Allopurinol is a drug used in the treatment of gout.
Gout is due to excessive production of uric acid. Xanthine
oxidase is an enzyme involved in the formation of uric acid from
hypoxanthine.
Allopurinol is a structural analog of hypoxanthine and hence it
is an antimetabolite of hypoxanthine.
Example 2: Anticancer drugs
Competitive inhibitors used in the treatment of cancer are
aminopterin and amethopterin (methotrexate).
They are structural analog of folic acid. They are competitive
inhibitors for the enzyme dihydrofolate reductase.
They are used in the treatment of leukaemia, a type of cancer. 29
b. Non-competitive inhibitors
Inhibitor binds to a site other than the active site
Binding causes a change in the structure of the enzyme so that it
cannot catalyze a reaction
The effects of non-competitive inhibition cannot be overcome by
increasing [S]
These inhibitors bind reversibly either to the free enzyme or the
ES complex to form the inactive complexes EI and ESI
The E-I complex formation does not affect the binding of
substrates but E-I-S complexes do not proceed to form products
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This kind of inhibition is common in allosteric enzymes which
have allosteric sites other than the substrate binding site
Example: the inhibition of L-threonine dehydratase by L-isoleucine
Reversible non-competitive inhibitors are rare.
Most of the known non-competitive inhibitors are irreversible.
They are referred as enzyme poisons.
1.Iodoacetate- blocks the formation of 1,3-bisphosphoglycerate
from glyceraldehyde-3-phosphate by inhibiting enzyme
glyceraldehyde-3-phosphate dehydrogenase
2. Fluoride- blocks the action of enolase, which converts 2-
phosphoglycerate to phosphoenolpyruvate.
3. Heavy metals like Hg2+, Ag+, Pb2+ and Arsenite are also enzyme
poisons. They interact with –SH group of enzyme and inactivate
it
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C. Uncompetitive reversible inhibition
• In this case the uncompetitive inhibitor binds only to ES complex
at locations other than the catalytic site (active site)
• Substrate binding modifies enzyme structure making inhibitor
binding site available
• 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.
• Inhibitor has no structural similarity to the substrate and does not
bind the free enzyme but binds the enzyme after complexation
with the substrate that exposes the inhibitor binding site (ESI).
• Its binding, although away from the active site, causes structural
distortion of the active site of the complexed enzyme that
inactivates catalysis.
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E + ES E +
S P
+
I
Uncompetitive inhibition
ESI
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Regulation of enzyme activity
Maintenance of an ordered state in a timely fashion and without
wasting resource
Regulation means to make an enzyme more or less active
Regulation is necessary to control the rates of reactions
1. Feedback/end product regulation
an enzyme early on in the pathway is inhibited by an end-
product of the metabolic pathway in which it is involved.
dephosphorylation,Methylation-demethylation,glycosylation etc
For example, phosphorylation of glycogen phosphorylase (an
enzyme that degrades glycogen) increases activity, whereas the
addition of phosphate to glycogen synthase (an enzyme that
synthesizes glycogen) decreases activity
3. Proteolytic cleavage
Zymogens (inactive precursor to an enzyme)
activated by cleavage of a specific peptide bond
active
In the small intestine, where their digestive properties
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Clinical Application of Enzymes
1. Use of enzymes in therapy
Coagulation Factors (II, VII, VIII, IX, X)- to Prevent
bleeding
DNase- to remove DNA clogging in lumen of tubes in cystic
fibrosis
Asparaginase: To remove Asn from extracellular fluid and
starve neoplastic cells
Patients who have had a heart attack or stroke are frequently
treated by intravenous administration of tissue plasminogen
activator (tPA) or streptokinase, enzymes that break down fibrin
clots that clog blood vessels
2. Evaluate the status of a given organ (liver, heart,) by serum
enzyme
3.To use enzymes as reagents to quantify the amount of an analyte
eg. Glucose oxidase as Glucose titer, uricase used for uric 37acid
analysis
Plasma enzymes
Plasma enzymes are of two sources
A. Plasma-derived /Plasma specific/ plasma functional enzymes
Their field of activity is plasma components and their activity is
higher in plasma than in cells, e.g. coagulation and lipoprotein-
metabolizing enzymes.
B. Cell-derived /Non-plasma specific /plasma non-functional
enzymes
A very low plasma level normally exists due to normal wear
and tear and diffusion through undamaged cell membranes.
Gross damage to the cells or abnormal membrane permeability,
overproduction of the enzymes or abnormal high cellular
proliferation and/or wear and tear may allow their leakage in
abnormally high amount into plasma.
Enzymes that have been shown to have diagnostic value are
present in plasma. 38
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Enzymes of different origin are present in plasma and are used
for diagnosis.
Because of normal cell turn over they present in small quantity ,
however, increased quantity is due cells injuries.
1. Acid phosphatase: Tumor marker in prostate carcinoma
2.Alanine aminotransferase(ALT): indicator of hepatocellular
damage.
3. Alkaline phosphatase: increase in cholestatic liver disease and
is marker of osteoblast activity in bone disease.
4. Amylase/Lipase: indicator of cell damage in acute pancreatitis.
5. Aspartate amino transferase (AST): indicator of hepatocellular
damage or as a marker of muscle damage such as MI
6. Creatine Kinase(CK): marker of muscle damage and acute MI.
7. γ-glutamyl transpeptidase: sensitive marker of liver cell
damage
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8. Lactate dehydrogenase: marker of muscle cell damage.
Application of enzymes in diagnosis of diseases
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Assignment
1. Write the Diagnostic function of enzymes and give
some examples
2. Drive a Michael-Menten equation
3. What are Isozymes? Give some examples with their
locaton and function.
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