[TRANS] LECTURE 4: ENZYMES

● Type of Reaction Catalyzed:

ENZYMES o oxidation of a substrate
● increase the rate of reactions without being changed in overall o reduction of a substrate
o introduction of double bond (oxidation)
process
o Catalyze nearly all chemical reactions taking place in the 1. TRANSFERASES
cells of the body ● Selected subclasses:
▪ Are compound or biological polymer, usually a protein, o Transaminases
that acts as a catalyst for a biochemical reaction o Kinases
o Not altered or consumed during reaction ● Type of Reaction Catalyzed:
o Reusable o transfer of an amino group between substrates
o selectively channel reactants (called substrates) into useful o transfer of a phosphate group between substrates
pathways 2. HYDROLASES
▪ Reactions in the body are mediated by enzymes ● Selected subclasses:
(Greek word en “in” and zyme “yeast”) o Lipases
o Proteases
o Nucleases
o Carbohydrases
o Phosphatases
● Type of Reaction Catalyzed:
o hydrolysis of ester linkages in lipids
o hydrolysis of amide linkages in proteins
o hydrolysis of sugar–phosphate ester bonds in nucleic acids
o hydrolysis of glycosidic bonds in carbohydrates
o hydrolysis of phosphate–ester bonds
3. LYASES
● Selected subclasses:
o dehydratases
o decarboxylases
o deaminases
o Hydratases
● Type of Reaction Catalyzed:
o removal of H2O from a substrate
o removal of CO2 from a substrate
o removal of NH3 from a substrate
o addition of H2O to a substrate
4. ISOMERASES
● Selected subclasses:
I. NOMENCLATURE o Racemases
A. Recommended name - commonly used enzyme names have o Mutases
the suffix “-ase” attached to the substrate of the reaction or to a ● Type of Reaction Catalyzed:
description of the action performed o conversion of D isomer to L isomer,or vice versa
B. Systematic name - the systematic naming system, enzymes o transfer of a functional group from one position to another in
are divided into six major classes the same molecule
o are unambiguous and informative 5. LIGASES
● Selected subclasses:
CLASSIFICATION OF ENZYME: IUB o Synthetases
1. OXIDOREDUCTASE - enzyme that catalyzes an oxidation- o Carboxylases
reduction reaction (browning reaction) ● Type of Reaction Catalyzed:
o Ex. Lactate dehydrogenase o formation of new bond between two substrates, with
▪ Organic Oxidation rxn - oxidation that increases participation of ATP
number of C-O bonds or decreases the number of C-H o formation of new bond bet a substrate and CO2, with
bonds participation of ATP
▪ Organic Reduction rxn - reduction that decreases
number of C-O bonds and or increases number of C-H II. PROPERTIES OF ENZYME
bonds ● Enzymes are protein catalysts that increase the velocity of a
2. TRANSFERASES - enzyme that catalyzes transfer of a chemical reaction, and are NOT consumed during the reaction
functional group other than hydrogen o RNAs with catalytic activity are called ribozymes, and are
o Ex. Transaminases and Kinases much less commonly encountered than protein catalysts
3. HYDROLASE - hydrolysis reaction which the addition of water o Protein Structure - scaffold to support and position active
molecule to bond causes bond to break site
4. LYASE - addition of group to a double bond or removal of a o Substrate - reactant in enzyme-catalysed reaction
group to form a double bond ▪ Enzyme acts to produce chemical reaction
o Ex. Dehydratase effects the removal of the components of
water from a double bond, Hydratase effects the addition of A. ACTIVE SITE - a special pocket or cleft
the components of water to a double bond o the region of enzyme where substrate molecules bind
5. ISOMERASE - isomerization (rearrangement of atoms) o most important part of enzyme as it directly catalyzes
o Only one reactant and one product in reactions where chemical reaction
isomerases are operative\ 1. Binding site - bonds and orient the substrate
6. LIGASE or SYNTHETASE - bonding together of two molecules 2. Catalytic site - catalyze a reaction of that substrate
into one with participation of ATP o Reduce chemical activation energy
● Enzyme–substrate complex - formed when a substrate binds to
CLASSIFICATION OF ENZYME BASED ON THE REACTION BEING the active site of enzyme
CATALYZED OXIDOREDUCTASES
● Selected subclasses: FACTORS AFFECTING ENZYMATIC ACTIVITY
o Oxidases 1. Temperature
o Reductases 2. pH
o Dehydrogenases

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3. Substrate Concentration
4. Enzyme Concentration E. Regulation - Enzyme activity can be regulated
F. Location within the cell - localized in specific organelles within the
2 TYPES OF ESC OR MODELS OF ENZYME ACTIVITY cell
1. Lock and key model - the active site in the enzyme has a fixed,
rigid geometrical conformation. III. HOW ENZYMES WORK (MECHANISM OF ACTION)
2. Induced fit model- allows for small changes in the shape or ● Two different perspectives:
geometry of the active site 1. First, catalysis in terms of energy changes that occur during
o Result to the enzymes flexibility the reaction, that is, enzymes provide an alternate,
energetically favorable reaction pathway different from the
uncatalyzed reaction.
2. Second, describes how the active site chemically facilitates
catalysis.
A. Energy changes occurring during the reaction (A ↔ T * ↔ B)
1. Free energy of activation: Because of the high, the rates of
uncatalyzed chemical reactions are often slow.
o energy difference between reactants and high-energy
intermediate
2. Rate of reaction: For molecules to react, they must contain
sufficient energy
o the lower the free energy of activation, the more molecules
have sufficient energy to pass through the transition state
3. Alternate reaction pathway: An enzyme allows reaction to
proceed under conditions prevailing the cell by providing alternate
reaction pathway with lower free energy of activation
B. Chemistry of the active site - complex molecular machine
employing a diversity of chemical mechanisms to facilitate
● Factors responsible for catalytic efficiency of enzymes:
1. Transition-state stabilization: stabilizing the transition state,
2. Catalysis: provide catalytic groups that enhance the probability
that transition state is formed.
o Chymotrypsin - enzyme of protein digestion in the intestine
3. Visualization of the transition state: product can be
visualized as being similar to removing a sweater from an
uncooperative infant

IV. FACTORS AFFECTING ENZYMATIC ACTIVITY

● Describes factors that influence reaction velocity of enzymes.
o Enzymic responses to these factors give us valuable clues
B. CATALYTIC EFFICIENCY- Enzyme-catalyzed reactions are highly
as to how enzymes function in living cells (that is, in vivo)
efficient, proceeding from 103–108 times faster than
A. Substrate concentration
uncatalyzed reactions.
1. Maximal velocity: Rate of an enzyme-catalyzed reaction
C. SPECIFICITY- Enzymes are highly specific, interacting with one or
increases with substrate concentration until maximal velocity
a few substrates and catalyzing only one type of chemical reaction
(Vmax) is reached
4 TYPES OF SPECIFICITY
o Rate or velocity of a reaction (v) - number of substrate
1. ABSOLUTE - enzyme will catalyze only one reaction
molecules converted to product per unit time
o Ex. Catalase enzyme catalyzes only Hydrogen peroxide
o Velocity - expressed as μmol of product formed per minute
(H2O2) to H2O and O2
o Saturation - all enzyme active sites were fully occupied and
2. GROUP - act only on molecules that have a specific functional
so reaction rate remains constant
group, such as hydroxyl, amino, or phosphate groups
2. Hyperbolic shape of the enzyme kinetics curve: Most
o Ex. Carboxypeptidase is group-specific; it cleaves amino
enzymes show Michaelis-Menten kinetics
acids, one at a time, from the carboxyl end of a peptide
o Hyperbolic - similar in shape to that of the oxygen-
chain.
dissociation curve of myoglobin
3. LINKAGE - act on a particular type of chemical bond
B. Temperature
o Ex. Phosphatases
1. Increase of velocity with temperature: Reaction velocity
4. STEREOCHEMICAL - act on a particular stereoisomer
increases with temperature until a peak/maximum velocity is
reached
D. Structural class, Holoenzymes, apoenzymes, cofactors, and
o Optimum temperature - enzyme exhibits maximum activity
coenzymes
2. Decrease of velocity with higher temperature: elevation of the
temperature results in decrease reaction velocity a result of
● Enzymes two general structural classes:
temperature-induced denaturation of enzyme
1. Simple enzymes - composed only of protein (amino acid chains)
o The optimum temperature for most human enzymes is
2. Conjugated enzymes - has a nonprotein part in addition to a
between 35 and 40°C.
protein part
C. pH
● Holoenzyme - the active enzyme with its nonprotein component
1. Effect of pH on the ionization of the active site: concentration
● Apoenzyme - protein part of the conjugated enzyme, inactive of H+ affects reaction velocity in several ways.
● Cofactor - If the nonprotein moiety is a metal ion such as Zn2+ 2. Effect of pH on enzyme denaturation: Extremes of pH can
or Fe2+ also lead to denaturation of the enzyme
● Coenzyme - small organic molecule 3. The pH optimum varies for different enzymes: pH at which
● Cosubstrates - only transiently associate with the enzyme maximal enzyme activity is achieved is different for different
● Prosthetic group - If the coenzyme is permanently associated enzymes, and often reflects optimum pH.
with the enzyme and returned to its original form (Ex. FAD) D. Enzyme concentration - concentration of substrate in a reaction
is much higher than that of the enzyme
Apoenzyme + nonprotein moiety (cofactor) = holoenzyme

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V. MICHAELIS-MENTEN EQUATION 1. Effect on Vmax: The effect of a competitive inhibitor is

reversed by increasing [S].
A. Reaction model - Leonor Michaelis and Maude Menten
proposed a simple model that accounts for most of the features of 2. Effect on Km: A competitive inhibitor increases the
enzyme-catalyzed reactions. apparent Km for a given substrate. Means that, in the
presence of a competitive inhibitor, more substrate is
needed to achieve 1⁄2Vmax.
3. Effect on the Lineweaver-Burk plot: Competitive inhibition
shows a characteristic Lineweaver Burk plot in which the
plots of the inhibited and uninhibited reactions intersect on
Where: the y-axis at 1/Vmax (Vmax is unchanged).
4. Statin drugs as examples of competitive inhibitors: This
S is the substrate
E is the enzyme group of antihyperlipidemic agents competitively inhibits the
ES is the enzyme–substrate complex first committed step in cholesterol synthesis.
B. Noncompetitive inhibition - recognized by its characteristic
P is the product
k1, k-1, and k2 are rate constants effect on Vmax
o occurs when the inhibitor and substrate bind at different sites
on the enzyme.
B. Michaelis-Menten equation - describes how reaction velocity
varies with substrate concentration: 1. Effect on Vmax: Noncompetitive inhibition cannot be
overcome by increasing the concentration of substrate.
Thus, noncompetitive inhibitors decrease the apparent
Vmax of the reaction.
2. Effect on Km: Noncompetitive inhibitors do not interfere
with the binding of substrate to enzyme. Km is unchanged.
3. Effect on Lineweaver-Burk plot: Noncompetitive
inhibition - readily differentiated from competitive inhibition
by plotting 1/vo versus 1/[S] and noting that the apparent
o Assumptions in deriving the MichaelisMenten rate Vmax decreases in the presence of a noncompetitive
equation: inhibitor, whereas Km is unchanged
C. Irreversible Inhibition
1. Relative concentrations of E and S: The concentration of
substrate ([S]) is much greater than the concentration of enzyme o Irreversible enzyme inhibitor - a molecule that inactivates
([E]), so that the percentage of total substrate bound by the enzymes by forming a strong covalent bond to an amino acid
D. Enzyme inhibitors as drugs - half of the ten most commonly
enzyme at any one time is small.
2. Steady-state assumption: [ES] does not change with time (the dispensed drugs in the United States act as enzyme inhibitors
steady-state assumption), that is, the rate of formation of ES is ● Aspirin - a non-prescription drug irreversibly inhibits
equal to that of the breakdown of ES (to E + S and to E + P). prostaglandins and thromboxane synthesis by inhibiting
3. Initial velocity: Initial reaction velocities (vo) are used in the cyclooxygenase.
analysis of enzyme reactions.
o means that the rate of the reaction is measured as soon as VII. ENZYME REGULATION
enzyme and substrate are mixed A. Allosteric enzymes - regulated by molecules called effectors
C. Important conclusions about Michaelis-Menten kinetics (also called modifiers) that bind noncovalently at a site other than
1. Characteristics of Km: Km - the Michaelis constant the active site.
o reflects the affinity of the enzyme for that substrate. B. Regulation of enzymes by covalent modification - Many
A. Small Km: small (low) Km reflects a high affinity of the enzyme enzymes may be regulated by covalent modification, most
for substrate frequently by the addition or removal of phosphate groups from
B. Large Km: large (high) Km reflects a low affinity of enzyme for specific serine, threonine, or tyrosine residues of the enzyme.
substrate because a high concentration of substrate is needed to 1. Phosphorylation and dephosphorylation:
half-saturate the enzyme Phosphorylation reactions - catalyzed by a family of
enzymes called protein kinases - use adenosine
2. Relationship of velocity to enzyme concentration: The rate of triphosphate (ATP) as a phosphate donor.
the reaction is directly proportional to the enzyme concentration at all o Phosphate groups- cleaved from phosphorylated enzymes
substrate concentrations. by the action of phosphoprotein phosphatases
3. Order of reaction: When [S] is much less than Km, the velocity of 2. Response of enzyme to phosphorylation: the
the reaction is approximately proportional to the substrate phosphorylated form may be more or less active than the
concentration. unphosphorylated enzyme.
o The rate of reaction is then said to be first order with C. Induction and repression of enzyme synthesis - Cells can
respect to substrate. also regulate the amount of enzyme present by altering the rate
o When [S] is much greater than Km, the velocity is constant of enzyme degradation or, more typically, the rate of enzyme
and equal to Vmax. synthesis.
D. Lineweaver-Burk plot o increase (induction) or decrease (repression) of enzyme
1. The equation describing the Lineweaver-Burk plot is: synthesis leads to an alteration in the total population of
active sites

D. Proteolytic enzyme and zymogen : regulating cellular enzyme

activity based on the production of enzymes in an inactive form.
o These inactive enzyme precursors are then “turned on” at
VI. INHIBITION OF ENZYME ACTIVITY
● Inhibitor - substance that can diminish the velocity of an enzyme-
catalyzed reaction
o irreversible inhibitors bind to enzymes through covalent
bonds
o reversible inhibitors bind to enzymes through noncovalent
bonds
A. Competitive inhibition - occurs when inhibitor binds reversibly
to the same site

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the appropriate time. o Serum amylase levels are most often used in the diagnosis
o Digestive enzymes needs activator like HCl so that of acute pancreatitis
zymogen are converted to proteolytic enzyme
1. Proteolytic enzyme: an enzyme that catalyzes the breaking of
peptide bonds that maintain the primary structure of a protein
2. Zymogen or proenzyme: inactive precursor of a proteolytic
enzyme
● EXAMPLES:

ZYMOGEN PROTEOLYTIC ENZYME

Pepsinogen pepsin
Tyrpsinogen trypsin
Chymotrypsinogen chymotrypsin
Angiotensinogen angiotensin CATALASE TEST
● Catalase - enzyme produced by microorganisms that live in
VIII. ENZYMES IN CLINICAL DIAGNOSIS oxygenated environments to neutralize toxic forms of oxygen
Plasma enzymes can be classified into two major groups. metabolites; H2O2.
1. Small group of enzymes - actively secreted into the blood by o enzyme in the liver that breaks down harmful hydrogen
certain cell types peroxide into oxygen and water.
2. Large number of enzyme - species released from cells during o When this reaction occurs, oxygen gas bubbles escape
normal cell turnover and create foam
● PAPAIN - known as papaya proteinase I - a cysteine protease
BIOCHEMICALLY IMPORTANT ENZYMES: enzyme present in papaya (Carica papaya) and mountain papaya
1. CREATINE KINASE: an enzyme that is found primarily in (Vasconcellea cundinamarcensis).
skeletal and cardiac muscle and in smaller fractions in the brain o Proteolytic enzyme
● TYPES OF CK: o Cystein protease enzyme
o muscle (CK-MM), brain (CK-BB), ● USES OF PAPAIN:
o cardiac tissue (CK-MB) - important marker in the diagnosis o remove extra fluid following trauma and surgery.
of acute myocardial infarction (AMI) o help with digestion, to treat parasitic worms, inflammation of
2. CARDIAC TROPONIN the throat and pharynx, shingles
● Description o Treat side effects of radiation therapy, or it may be used in
o Troponin I and T are sensitive markers of cardiac injury combination with other therapies to treat tumors
o Troponin I is found solely in the cardiac muscle, and o treat insect or animal bites, infected wounds, sores, and
o Troponin T is found in both cardiac and skeletal muscle ulcers.
● Clinical Significance ● SYMPTOMS:
o Troponin levels begin to rise within 4 hours of onset of chest o sore muscles
pain. Levels should be drawn on admission and within 8 to o Diarrhea
12 hours thereafter. Patients with elevated troponin levels o hay fever
are considered at high risk for a significant cardiac event. o runny nose
3. GASTROINTESTINAL TESTS o psoriasis
A. Alanine Aminotransferase/ serum glutamic pyruvic
transaminase (SGPT) ● Simple enzyme - composed only of protein not bound to any
i. liver tissue. It is also located in myocardial, muscle, nonproteins
and renal tissue ● Conjugated enzyme - has a nonprotein part in addition to a
ii. considered a specific marker for liver disease protein part
B. Aspartate Aminotransferase/ serum glutamic oxaloacetic ● Apoenzyme - protein part of a conjugated enzyme
transaminase (SGOT) ● Cofactor - inonprotein part of a conjugated enzyme
i. found in the liver. It is also present in the heart, kidney, ● Prosthetic group - Tightly bound cofactor to the apoenzyme
pancreas, lungs, and skeletal muscle ● Holoenzyme - biochemically active conjugated enzyme produced
ii. For diagnosis of liver disease from an apoenzyme and a cofactor
C. C. g-Glutamyl Transpeptidase ● Coenzyme or cosubstrate - is a small organic molecule that
i. enzyme found in the liver, kidney, and pancreas. GGT serves as a cofactor in a conjugated enzyme
levels are useful in the diagnosis and monitoring of ● Activator - the inorganic cofactor
alcoholic liver disease ● Substrate (S) - is the reactant in an enzyme-catalyzed reaction
ii. Increased GGT may be seen in alcoholic liver disease, ● Product (P) - the biomolecules formed by enzyme mediated
metastatic liver disease, obstructive jaundice, reactions
cholelithiasis, and pancreatitis ● metal-activated enzymes - Enzymes that require a metal ion
D. Lactate Dehydrogenase - enzyme involved in the cofactor
interconversion of lactate and pyruvate. ● Metalloenzymes - enzymes that contain tightly bound metal ions
o is found in many tissues, including heart, brain, liver, skeletal
muscle, kidneys, lungs, and RBCs. MEDICAL USES OF ENZYMES
o LDH4 and LDH5 are present in liver tissue, and elevations LIPASE:
may be seen in liver disease such as hepatitis and cirrhosis. ● an enzyme catalyzing the hydrolysis of fats. It is secreted by
o LDH1 and LDH2 may be useful in the diagnosis of pancreas and Liver
myocardial infarction ● The plasma lipase level may be low in liver disease, Vitamin A
E. Lipase deficiency, some malignancies, and diabetes mellitus.
o enzyme that aids in the digestion of fat. It is primarily o It may be elevated in acute pancreatitis and pancreatic
secreted by the pancreas. carcinoma.
o useful in the diagnosis of pancreatitis and is considered a
more specific marker for acute pancreatitis than amylase
Α- AMYLASE
F. Amylase
o enzyme that aids in digestion by breaking down complex ● α- amylase is the enzyme concerned with the break down of
carbohydrates into simple sugars. dietary starch and glycogen to maltose.
o produced in the pancreas and salivary glands

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o Ipresent in pancreatic juice and saliva as well as in liver

fallopian tubes and muscles
▪ The enzyme is excreted in the Urine.
▪ main use of amylase estimations is in the diagnosis of
acute pancreatitis

TRYPSIN
● secreted by pancreas
o Elevated levels of trypsin in plasma occur during acute
pancreatic disease.

ALKALINE PHOSPHATES (ALP)

● are a group of enzymes, which hydrolyze phosphate esters at an
alkaline pH
o found in bone, liver, kidney, intestinal wall, lactating
mammary gland and placenta.

ACID PHOSPHATASE (ACP)

● catalyzing the hydrolysis of various phosphate esters at acidic pH
is found in the prostate, liver, red cells, platelets and bone
o It may be elevated in metastatic prostatic carcinoma.

TRANSAMINASES
● Two transaminases are of clinical interest.
1. Aspartate Transaminase, AST ( Glutamate oxaloacetate
transaminase, GOT ) catalyzes the transfer of the amino group of
aspartic acid to α- ketoglutarate forming glutamate and oxaloacetate.
AST or GOT is widely distributed, with high concentration, in the heart,
liver, skeletal muscle, kidney and erythrocytes, and damage to any of
these tissues may cause raised levels.
2. Alanine transaminase, ALT (Glutamate pyruvate transaminase,
GPT ) Transfer the amino group of alanine to α- ketoglutarate, forming
glutamate and pyruvate. It is present in high concentration in liver and
to a lesser extent in skeletal muscle, kidney and heart.

LACTATE DEHYDROGENASE (LDH)

● catalyzes the reversible interconversion of lactate and pyruvate
o widely distributed with high concentrations in the heart,
skeletal muscle, liver, kidney, brain and erythrocytes.
o
● Creatine kinase (CK) or creatine phosphokinase (CPK) - found
in heart muscle brain and skeletal muscle. Measurement of serum
creatine phosphokinase activity is of value in the diagnosis of
disorders affecting skeletal and cardiac muscle. The level of CPK
in plasma highly increased in myocardial infarction.

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