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ENZYMES o coenzyme – organic

compound cofactor that
- catalytic proteins allows maximum catalytic
- digest food, contract muscles, power
synthesize hemoglobin
- catalyze reaction that supply
necessary biomolecules and energy Protein (apoenzyme) + Cofactor =
for survival Active Enzyme (holoenzyme)
- Catalyst – increases reaction rate by
changing the way a reaction takes
place without undergoing any net • Apoenzyme – inactive
change itself. protein portion of the enzyme
- lowers activation energy for • Holoenzyme – active
chemical reactions (i.e., less energy enzyme (apoenzyme +
required which leads to increase in cofactor)
rate of reaction) • Cofactors – inorganic metal
ions; organic compounds
Chemical reaction in cells derived from B vitamins
(coenzymes)
- Incredibly fast
- pH 7.4 METAL IONS (COENZYMES)
- body temp: 37°C
VITAMINS
Carbonic Anhydrase
1. Thiamine (B1)
- enzyme in the blood o Coenzyme Derivative:
- converts carbon dioxide and water to Thiamine Pyrophosphate
carbonic acid (TPP)
- chemical reaction occurs 10 million o Significance: removal of
times as rapidly as it does in the CO2, decarboxylation
absence of carbonic anhydrase reactions
- 1 molecule can hydrate about 1
million molecules of CO2/second
2. Riboflavin (B2)
TYPES OF ENZYMES o Coenzyme Derivative:
Flavin adenine dinucleotide
1. Simple proteins (FAD) and Flavin
o consists only of a polypeptide mononucleotide (FMN)
chain o Significance: in coenzymes
o tertiary protein structure of FAD and FMN
simple enzyme
2. Conjugated proteins 3. Niacin (B3, nicotinic acid,
o contains a cofactor – nicotinamide)
nonprotein portion of enzyme o Coenzyme Derivative:
(e.g., metal ion) Nicotinamide adenine
dinucleotide (NAD) and
nicotinamide adenine
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dinucleotide phosphate Enzyme: cytochrome oxidase,

(NADP) catalase, dehydrogenases
o Significance: in coenzyme
NAD
4. Mg2+
4. Pyridoxine (B6) Enzyme: pyruvate kinase
o Coenzyme Derivative:
Pyridoxal phosphate (PP)
o Significance: in coenzyme 5. Mn2+
for amino acid and lipid Enzyme: arginase, pyruvate
metabolism carboxylase, phosphatases, succinic
dehydrogenase, glycosyl
5. Folic Acid (B-C) transferases, cholinesterase
o Coenzyme Derivative:
Tetrahydrofolate (THF) 6. K+
o Significance: in coenzyme Enzyme: pyruvate kinase
for amino acid and nucleic
acid metabolism 7. Zn2+
Enzyme: carbonic anhydrase,
6. Biotin carboxy peptidase, lactic
o Coenzyme Derivative: N/A dehydrogenase, alcohol
o Significance: in coenzyme dehydrogenase
for carboxylation reactions

7. Pantothenic acid (B5)

o Coenzyme Derivative: NAMES AND CLASSIFICATION OF ENZYMES
Coenzyme A (CoA) - describe the compound or reaction
o Significance: in coenzyme A that is catalyzed
- suffix: -ase
8. Ascorbic Acid (Vitamin C)
o Coenzyme Derivative: N/A Oxidase – catalyzes an oxidation reaction
o Significance: coenzyme;
Dehydrogenase – removes hydrogen
delivers hydride ions;
atoms
antioxidant
Sucrase – hydrolyzes glycosidic bond in
METAL IONS sucrose
1. Ca2+
Enzyme: thromboplastin Lipase – hydrolyzes ester bonds in lipids

2. Cu2+
Enzyme: tyrosinase, cytochrome Some early known enzymes (suffix: -in):
oxidase
Papain – found in papaya
3. Fe2+, Fe3+ Rennin – found in milk
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Pepsin and trypsin – catalyze hydrolysis of o

General reaction: rearrange
proteins the atoms of a substrate;
transfer of groups within
molecules to yield isomeric
6 MAIN CLASSES OF ENZYMES forms
o Examples: isomerases,
1. Oxidoreductases epimerases
o General reaction: o Reactions catalyzed:
oxidation/reduction convert cis to trans; ketose to
o Examples: oxidases, aldose; convert D- to L-
dehydrogenases, reductases isomer
o Reactions catalyzed: 6. Ligases
oxidation, remove hydrogen, o General reaction: synthesis
add hydrogen of larger molecules;
2. Transferases formation of C-C, C-S, C-O,
o General reaction: move a and C-N bonds by
functional group condensation reactions
o Examples: transaminases, coupled to ATP cleavage
kinases o Examples: synthetases,
o Reactions catalyzed: carboxylases
transfer of amino groups o Reactions catalyzed:
3. Hydrolases combine two molecules, add
o General reaction: hydrolysis CO2 to a substrate
of bonds (transfer of
functional groups to water) ENZYME-SUBSTRATE COMPLEX
o Examples: peptidases,
lipases, amylase, Substrates – small group of reacting
phosphatase molecules
o Reactions catalyzed: Steps in an enzyme catalyzed reaction:
hydrolyze peptide bonds,
hydrolyze ester bonds in
triglycerides, split 1,4-
glycosidic bonds in amylose,
hydrolyze phosphate groups
4. Lyases
o General reaction: remove The first step involves the encounter of the
small groups to form double enzyme with its substrate and the formation
bond; add small groups to of an enzyme-substrate complex.
double bonds
o Examples: decarboxylases,
dehydrases, deaminases Active site – part of the enzyme that binds
o Reactions catalyzed: with the substrate
remove CO2, remove H20,
remove NH3 Characteristics of enzyme active site:
5. Isomerases
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1. Enzyme active sites are pockets or
clefts in the surface of the enzyme
2. Its shape is complementary to the
shape of the substrate
3. An enzyme attracts and holds its
substrate by weak, noncovalent
interactions
4. Conformation of the active site
determines specificity of enzyme
because only the substrate that fits
into the active site will be used in a
reaction.
MODELS OF ENZYME ACTIVITY
- active site “molds” itself around
substrate
- shape of the active site conforms
precisely to the shape of the
substrate (it may also change)
SPECIFICITY OF ENZYME-SUBSTRATE COMPLEX
Enzyme-substrate interaction
requirement:
- surface of enzyme and substrate
must be complementary
Enzyme specificity – ability of an enzyme
to bind only one, or a very few, substrates
and thus only catalyze a single reaction.

Classes of Enzyme Specificity

Lock-and-key model of enzyme-substrate 1. Absolute specificity
binding (Figure A) o An enzyme that catalyzes the
reaction of only one
- Emil Fischer (1894) substrate (i.e., Aminoacyl
- substrate simply snapped into place tRNA synthetases)
like a key into a lock 2. Group specificity
- assumes that the enzyme active site o An enzyme that catalyzes
has a rigid structure that is precisely processes involving similar
complementary in shape and charge molecules containing the
distribution to the substrate. same functional group
o Hexokinase – a group-
specific enzyme that
Induced-fit model of enzyme-substrate catalyzes addition of a
binding (Figure B) phosphoryl group to the
hexose sugar glucose in the
- Daniel E. Koshland, Jr. (1958)
first step of glycolysis. It can
- enzyme is a flexible pocket that
also add a phosphoryl group
approximates the shape of the
to several other six-carbon
substrate
sugars.
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3. Linkage specificity structure; hence, a cell cannot

o An enzyme that catalyzes the survive very high temperatures.
formation or breakage of only
Autoclaving
certain bonds in a molecule
o Proteases (trypsin, o Works on the principle of
chymotrypsin, elastase) – pressure cooker: air is
enzymes that selectively pumped out of the chamber,
hydrolyze peptide bonds and steam under pressure is
(linkage specific) pumped into the chamber
until a pressure of two
4. Stereochemical specificity atmospheres is achieved.
o An enzyme that can o Most effective means of
distinguish one enantiomer destroying heat-resistant
from the other endospores formed by
o Most of the enzymes in the bacteria (genera Bacillus and
human body show Clostridium, which are
stereochemical specificity responsible for deadly
(we only use D -sugars and L diseases such as anthrax,
-amino acids, enzymes gas gangrene, tetanus,
involved in digestion and botulism food poisoning)
metabolism recognize only
those stereoisomers) Thermophiles

FACTORS AFFECTING ENZYME ACTIVITY o Live in environments where

temperatures range from
Temperature 50°-80°C

- The rate of the uncatalyzed reaction Ph

steadily increases (Figure a) with
- Most enzymes function at pH 7
increasing temperature because
Denaturation - enzyme loses its
more collisions occur with sufficient
biologically active configuration
energy to overcome the energy
barrier for the reaction.
pH optimum - pH at which an
- Temperature optimum – enzyme is
enzyme functions optimally
functioning optimally; rate of the
reaction is maximal. Pepsin – proteolytic digestive
- Above the temperature optimum, enzyme that effectively degrade
increasing temperature begins to proteins at pH 2 (pH optimum)
increase the vibrational energy of
the bonds within the enzyme. Trypsin – proteolytic enzyme found
Eventually, so many bonds and in the intestine.
weak interactions are disrupted that Both pepsin and trypsin cleave
the enzyme becomes denatured, peptide bonds by virtually identical
and the reaction stops. amino acids, yet their amino acid
- Heating enzymes and other proteins sequences have evolved so that
destroys their three-dimensional
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they are stable and active in very REGULATION OF ENZYME ACTIVITY

different environments.
Allosteric enzymes
Lysosome – called “suicide bags”
because they are membrane-bound - Enzymes that have more than one
vesicles containing about 50 binding sites
different kinds of hydrolases which - a metabolic pathway generally
degrade large biological molecules consists of a linear sequence of
into small molecules that are useful enzymes which process a given
for energy-harvesting reactions. (pH substrate through multiple
optimum: 4.8) transformations.

Enzymes of lysosome being Effector molecules – small

released into cytoplasm – molecules bound to allosteric
destruction of cellular enzymes which increases or
macromolecules & death of cell; decreases activity at another site
cytoplasmic pH 7.0-7.3 renders
lysosomes inactive o Positive modulators
(allosteric activators) –
stimulate binding at other
Substrate Concentration sites (increase affinity)
o Negative modulators
- For a given amount of enzyme, a
(allosteric inhibitors) – inhibit
reaction will go faster as the
binding at other sites
amount of substrate increases.
(decrease affinity)
However, when all the enzyme
molecules are combined with
The results can be:
substrate, a maximum reaction rate
o Negative allosterism –
is reached. At this point, the enzyme
effector binding converts
molecules are saturated, and there
active site to an inactive
can be no further increase in rate
configuration
even if more substrate is added.
o Positive allosterism –
effector binding converts
active site to the active
Enzyme Concentration configuration.
- In general, the concentration of o
substrate in a reaction is much Sub-unit interactions in allosteric
higher than that of the enzyme. As enzyme:
the enzyme concentration is
increased, the reaction rate
increases, because more substrate
molecules can undergo reaction.
- Increasing enzyme concentration
increases rate of rxn
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F will inhibit an earlier enzyme (Enz1) in

the pathway
– when enough F produced, it
blocks its own biosynthesis
o often the first enzyme of a
pathway is a regulatory one
(represents a 'committed'
step)

- Feedback inhibition is an effective

metabolic on-off switch

Glycolysis – main metabolic

pathway for breakdown of glucose to
produce ATP energy for cell (must
be responsive to demands of the
body.)
o In the third reaction,
phosphoryl group is
transferred from an ATP
molecule to a molecule of Proenzyme
fructose-6-phosphate
(catalyzed by enzyme: - Inactive form of enzyme
phosphofructokinase) - Converted by proteolysis (hydrolysis
- If energy demand is low, reactions of protein) to the active form when it
slow down has reached the site of its activity

Feedback Inhibition - Pepsinogen: inactive proenzyme in

- aka feedback regulation the stomach produced by cells that
- basis: allosteric enzymes also produce pepsin; has additional
42 amino acids

Starting material (A) is converted into B

by the enzyme (Enz1), so on until the
final product, F. Key metabolite (F)
produced is via a multi-enzyme
metabolic pathway; when [F] increases,
Protein Modification
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- Process where a chemical group is
covalently added to or removed from
the protein (either activates enzymes
or turns it off)
- Phosphorylation/dephosphorylati
on: enzyme regulation by reversible
covalent modification; common
strategy for cells to regulate their
metabolic activities
Protein kinases – add phosphoryl
(phosphorylation)
Phosphatases – remove phosphoryl
(dephosphorylation)
(note: this is a hydrolysis reaction)
- attachment of a phosphate group to
the side chain of a protein residue
(Ser, Thr, Tyr, His) introduces a
bulky, charged group that can
change the protein’s conformation
and/or activity
INHIBITION OF ENZYME ACTIVITY
Enzyme inhibitors – chemicals that either
eliminate or drastically reduce catalytic
ability of enzymes
Penicillin – inhibits several enzymes
involved in synthesis of bacterial cell walls.
Enzyme inhibitors are classified based on
whether the inhibition is reversible or
irreversible, competitive or noncompetitive.
Reversibility – whether the inhibitor will
eventually dissociate from the enzyme,
releasing it in active form.
Competition – whether inhibitor is a
structural analog or look-alike of the natural
substrate. If so, inhibitor and substrate will
compete for the enzyme active site.
Irreversible Inhibitors
- inhibitors bind very tightly
(sometimes covalently) to one of the
R groups of an amino acid in the
active site (e.g., arsenic)
- active site is permanently blocked,
enzyme is irreversibly inactivated
(enzyme-substrate complex cannot
form); eliminates catalysis by
interfering with catalytic groups of
the active site
- snake venoms and nerve gases,
cyanide
Reversible, Competitive Inhibitors
- structural analogs
- resemble structure and charge
distribution of the natural substrate
for an enzyme (because of this,
inhibitor can occupy enzyme active
site, however, no reaction can occur,
and enzyme activity is inhibited)
- both inhibitor and substrate compete
for binding to the enzyme active site
- Malonic acid: competitive inhibitor
of succinic dehydrogenase
Methanol poisoning (CH3OH)
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- Ethanol (CH3CH2OH): inhibitor;
structural analog of methanol
Sulfa drugs (antimicrobics)
- Competitive inhibitors of bacterial
enzyme needed for synthesis of
vitamin folic acid.
Folic acid – vitamin required for
transfer of methyl groups in
biosynthesis of methionine and
nitrogenous base required to make
DNA and RNA
o Without folic acid, growth of
bacteria is retarded, and
eventually kills them.
o PABA (para-aminobenzoic
acid) – precursor of folic
acid; substrate for an early
step in folic acid synthesis
▪ PABA and sulfa drugs
are structural
analogs, hence, they
will compete for the
enzyme active site of
bacteria
Reversible Noncompetitive Enzyme
Inhibitors
- bind to R groups of amino acids or to
the metal ion cofactors (not to the
active site, hence, it is
noncompetitive ang inhibitor)
- Inhibitor can alter the tertiary
structure of the enzyme, therefore,
alters/modifies the shape of the
active site (the same way that
allosteric effector does)
- Binding is weak, and enzyme activity
is restored when inhibitor dissociates
from enzyme-inhibitor complex
- Binding is nonspecific, therefore it
inactivates a broad range of
enzymes
USES OF ENZYMES IN MEDICINE
Analysis of the levels of enzymes
(concentrations) in the blood can provide
information about a patient’s medical
condition.
Enzyme assays – measures amount of
certain enzymes in the blood; very precise
and specific because they are based on
specificity of enzyme-substrate complex.
e.g.,
Myocardial infarction (heart attack)
- heart muscles destroyed and will
release enzymes in the bloodstream
- In acute myocardial infarction, three
cardiac biomarkers (creatine kinase-
MB, myoglobin, and troponin I) are
important
- SGPT and SGOT increase in
concentration
Inflammation of pancreas
- High levels of amylase and lipase in
blood
Liver diseases (e.g., cirrhosis and
hepatitis)
- High levels of SGPT (serum
glutamate-pyruvate transaminase) or
SGOT (serum glutamate-
oxaloacetate transaminase)
Blood Urea Nitrogen (BUN) test
- Clinical analysis of urea in blood
- If urea is converted to ammonia (thru
enzyme urease), ammonia becomes
an indicator of urea because it is
produced from urea and is easily
measured.
- Useful in diagnosis of kidney
malfunction and serves as one
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example of utility of enzymes in Acetylcholine – neurotransmitter

clinical chemistry. (chemical messenger) that transmits
a message from nerve cell to muscle
cell; stored in membrane-bound
ISOENZYMES bags (synaptic vesicles) in the nerve
cell ending.
- Forms of the same enzyme with 1. Arrival of nerve impulse at the end
slightly different amino acid plate of nerve axon causes an influx
sequences. of Ca2+
- Binding and catalytic sites are the 2. Acetylcholine containing vesicles
same but there are differences in the migrate to the nerve cell membrane
scaffolding of the enzyme that that is in contact with muscle cell
maintains the three-dimensional (presynaptic membrane)
structure (tertiary) of the proteins. 3. Vesicles fuse with presynaptic
- Differs in the amino acids found in membrane and release
the perimeter of the enzyme neurotransmitter
4. Acetylcholine diffuses across the
Creatine phosphokinase (CPK)
nerve synapse and binds to the
- Has three isoenzymes: acetylcholine receptor protein ® in
the postsynaptic membrane of the
CPK 1 – enzyme in the brain muscle cell
CPK 2 – enzyme in the heart 5. Receptor opens pores in the
membrane through which Na+ and
CPK 3 – enzyme in the skeletal muscle K+ ions flow into and out of the cell,
respectively.
6. The flow of ions generates nerve
Lactase dehydrogenase (LDH) impulse and causes muscles to
contract
- 5 isoenzymes 7. If acetylcholine remains at the
LDH 1 – in heart attack, concentration of junction, stimulation of muscle
LDH 1 increases (the rest, LDH 2 to contraction continues
LDH 5, remains the same) 8. To stop, acetylcholine should be
hydrolyzed, then it will be destroyed
by acetylcholinesterase. When this
happens, nerve stimulation ceases.
CHEMISTRY AT THE CRIME SCENE
Organophosphates
Enzymes, Nerve Agent, and Poisoning
- Most important inhibitors of
Acetylcholinesterase acetylcholinesterase.
- catalyzes the hydrolysis of the Sarin (isopropylmethylfluorophosphate)
chemical messenger, acetylcholine,
in the transmission of nerve - nerve agent that forms a covalently
impulses at the neuromuscular bonded intermediate with the active
junction site of acetylcholinesterase;
irreversible, noncompetitive inhibitor
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- covalently bonded intermediate is Reaction rate (Vmax) – dependent only on

stable, making acetylcholinesterase (Eo).
inactive (no longer able to break
NOTE: Km = [S] when enzyme is at half of
down acetylcholine) which results in
its velocity.
muscle spasm
Pyridine aldoxime methiodide (PAM)
- antidote for poisoning by
organophosphates
- displaces organophosphate group
from the active site of the enzyme,
thus alleviating effects of the poison

KINETICS OF ENZYME-CATALYZED REACTIONS

Consider a single substrate reaction:

For a given amount of enzyme, a reaction

will go faster as the amount of substrate
increases. However, when all the enzyme
molecules are combined with substrate, a Michaelis-Menten Equation:
maximum reaction rate is reached. At this
point, the enzyme molecules are saturated - Km: Michaelis-Menten constant;
and there can be no further increased in independent of enzyme
rate even if more substrate is added. concentration; is equal to the
substrate concentration when
reaction rate is half of its maximal
Rate of enzyme catalyzed reaction is value.
proportional to substrate concentration (S) - Equation relates Vo, Vmax, and S
only at low (S). As (S) is increased, reaction - (S): total substrate concentration at
rate falls off and no longer depends on (S). the start of rxn; under this condition,
(S) >> (Eo)
- Enzyme concentration is
incorporated in Km

DETERMINATION OF Km AND VMAX (Graphical

Graph of initial velocity against initial
substrate concentration at constant total Evaluation)
enzyme concentration for a single substrate 1. Lineweaver-Burk Equation
enzyme – catalyzed reaction. o Reciprocal form of Michaelis-
Menten equation
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2. Eadie-Hofstee Equation
o Michaelis-Menten equation is
algebraically rearranged into:

Y and x-intercepts correspond to Vmax and

Vmax/Km, respectively
3. Hanes Equation
o the Lineweaver-Burke
equation is multiplied
throughout by (So):

+I: with inhibitor

-I: without inhibitor
Effect of a Competitive Inhibitor on Vmax
and Km of an enzyme-catalyzed reaction:

Effect of a Noncompetitive Inhibitor on

Vmax and Km of an enzyme-catalyzed
reaction:
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+I: with inhibitor

-I: without inhibitor