Enzymes:

“Helper” Protein molecules

Regents Biology
 What Are Enzymes?
• Most enzymes are
Proteins (tertiary and
quaternary structures)

• Act as Catalyst to
accelerate a reaction

• Not permanently
changed in the
process

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 Characteristics of Enzymes
1. Lower the activation energy of a reaction.
- Activation energy is the energy needed to get a reaction
started.
2. Increases the rate of a reaction but does not
cause a reaction to occur that would not occur
in its absence.
- Enzymes are biological catalysts
2H2O2 -> 2H2O + O2 One molecule of catalase can break 40
million molecules of hydrogen peroxide each second.
3. Are not used up or changed by the reaction.
4. Exhibit specificity, competition, and saturation.
 Nomenclature and Classification
 Recommended name
(commonly used)

 substrate + -ase e.g.

sucrase, urease,
Oh, I get it!
glucosidase) They end in -ase
 Action performed e.g.
lactate dehydrogenase
and adenylyl cyclase

 Systematic name (IUBMB)

divided enzymes into 6
major classes.

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Systematic name (IUBMB)
divided enzymes into 6 major
classes:
Oxidoreductase, Transferase,
Hydrolases, Lyases,
Isomerases, Ligases
Class 1. Oxidoreductases- catalyze redox processes

Example:

Class 2. Transferases- transfer chemical groups

from one molecule to another or to another part
of the same molecule.

Example:
Class 3. Hydrolases- cleave a bond using water
to produce two molecules from one.
example: cleavage of a peptide bond

Class 4. Lyases- remove a group from or add a

group to double bonds.
Class 5. Isomerases- interconvert isomeric structures by
molecular rearrangements.

Class 6. Ligases -- join two separate molecules

by the formation of a new chemical bond usually
with energy supplied by the cleavage of an ATP.
example:
 Enzyme properties
A. The active site
 Enzymes contain special pocket or cleft that
binds the substrate
 One part of an enzyme, the active site, is
particularly important
 The shape and the chemical environment
inside the active site permit a chemical reaction
to proceed more easily
B. Catalytic efficiency:
highly efficient (103-108) faster than uncatalyzed reaction

C. Specificity:
Highly specific, catalyzes only one type of chemical reactions.
The substrate of an enzyme are the reactants that are
activated by the enzyme
Enzymes are specific to their substrates
The specificity is determined by the active site
 D. Holoenzymes:
• The enzyme with cofactor is called holoenzyme, the protein portion is
apoenzyme.
• The enzyme without cofactor doesn’t show biological activity
• Enzyme activity relies on binding of certain inorganic molecules before
substrate binding can occur. e.g. Ca2+ & Mg2+.
• Tightly bound cofactors are called prosthetic groups
• Coenzymes are the cofactors that are bound & released easily.
• Many vitamins are coenzymes
 Coenzymes
• Coenzymes are organic molecules.

• Do not alter the enzyme’s binding site.

• Accept electrons from an enzyme in one metabolic pathway and

transfer them to an enzyme in another.

• They are not needed in large amounts.

• The water soluble vitamins (thiamine (B1) niacin, riboflavin (B2)) are
all good examples of cofactors.
– NAD (nicotinamide adenine dinucleotide)
– FAD (flavine adenine dinucleotide)
– Coenzyme A (pantothenic acid)
 Coenzyme NAD+

• NAD+ transports electrons from one

metabolic pathway to another
nicotinamide-adenine dinucleotide (NAD),
nicotinamide-adenine dinucleotide phosphate
(NADP)
-- use = redox Coenzyme
reactions with
H transfer
-- its vitamin =
P
niacin or B3 =
nicotinamide
& nicotinic acid
 riboflavin
Coenzyme =
flavin mononucleotide
(FMN),
flavin adenine
dinucleotide (FAD)
both act as prosthetic groups
-- use = redox reactions
-- its vitamin = riboflavin or B2
Coenzyme = Coenzyme A (CoA)
-- use = activates carbonyl groups
and in acyl transfer
(acetyl- CoA, synthesis
of fats and steroids)
-- its vitamin = pantothenic acid -- disease= GI
problems, emotional instability, burning sensation
in extemities

acetyl
Acetyl CoA
E. Regulation:
can be activated or inhibited by different
substances.
F. Location within the cell: each
enzyme is localized in specific
organelle within the cell, which isolates
the reaction substrate or product from
other competing reactions.
How do enzymes Work?
Enzymes work by
weakening
bonds which
lowers
activation
energy

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 Factors affect Reaction velocity
A. Substrate concentration
 Maximal velocity:
rate of enzyme-catalyzed rxn  with [S]
until Vmax is reached  (saturation)

 Hyperbolic shape of the enzyme

kinetics curve:
Most enzymes show Michaelis-Menten
kinetics  plot of vo against [S] is
hyperbolic.
allosteric enzymes frequently show a
sigmoidal curve
 Effect of Enzyme or Substrate
Concentration on Reaction Rate
• The reaction rate is directly
related to enzyme
concentration.

• The reaction rate can reach

a maximum (Vmax) when
the enzyme is saturated.
 B. Temperature
 Increase of velocity with temperature:
• rxn velocity increases with temperature
until a peak velocity is reached.
 Decrease of velocity with higher
temperature:
• as a result of temperature-induced
denaturation of the enzyme
• Boiling =denature protein = unfold = lose
shape
Optimum temperature
• greatest number of collisions between
enzyme & substrate
• for most human enzymes: 35 - 40°C.
 C. pH
 changes in pH changes protein shape
 Effect of pH on the ionization of the active site:
Catalytic process requires that E & S have specific chemical groups in either an
ionized or unionized state in order to interact

 Effect of extremes of pH on enzyme denaturation:

because the structure of the catalytically active protein molecule depends on the
ionic character of the amino acid side chains.
 The pH optimum varies for different
enzymes:
pH at which maximal enzyme activity is
achieved is different for different enzymes
 reflects [H] at which enzyme functions in
body
• pepsin (stomach) = pH 3
• trypsin (small intestines) = pH 8
 Inhibition of enzyme activity
 Inhibitor: any substance that can diminish the velocity of an enzyme-
catalyzed reaction.
 Irreversible inhibition:
 bind to enzymes through covalent bonds
 occurs when an inhibited enzyme does not regain activity on
dilution of the enzyme-inhibitor complex.

 Reversible inhibition:
bind to enzymes through noncovalent bonds.
Dilution of enzyme-inhibitor complex results in dissociation of
the reversibly bound inhibitor, and recovery of enzyme activity
Reversible inhibitors can be competitive or noncompetitive.
 Competitive inhibition
 inhibitor competes with the substrate for the same site.
 Vmax unchanged  effect of competitive inhibitor is reversed by [S]
 Apparant Km is increased  more S is needed to achieve ½Vmax
 statin drugs are example
 Statin drugs as examples of competitive inhibitors

Lovastatin competes
with HMG-CoA for the
active site of HMG-CoA
reductase

inhibit de novo
cholesterol synthesis,
thereby lowering plasma
cholesterol levels
 Noncompetitive Inhibition
• Does not have a structure like substrate

• Binds to the enzyme but not active site

• Changes the shape of enzyme and active site

• It can bind either free E or the ES complex

• S cannot fit altered active site

• No reaction occurs

• Effect is not reversed by adding substrate

 Noncompetitive inhibition
 Km is unchanged
 Vmax is decreased
• e.g. Lead forms covalent bonds with the sulfhydryl side chains of
cysteine in proteins. Ferrochelatase (catalyzes the insertion of
Fe2+ into protoporphyrin) is sensitive to inhibition by lead.
• certain insecticides, whose neurotoxic effects are a result of their
irreversible binding at the catalytic site of acetylcholinesterase
(an enzyme that cleaves the neurotransmitter, acetylcholine).
 Enzyme inhibitors as drugs

 β-lactam antibiotics (e.g. penicillin & amoxicillin) act by

inhibiting enzymes involved in bacterial cell wall synthesis.

 Angiotensin-converting enzyme (ACE) inhibitors (e.g.

captopril, enalapril, and lisinopril) lower blood pressure by
blocking the enzyme that cleaves angiotensin I to form the
potent vasoconstrictor, angiotensin II.
 Regulation of enzyme activity
(Allosteric regulation)
 Allosteric enzymes, which frequently catalyze
the committed step early in a pathway, are
regulated by effectors (or modifiers) that bind
noncovalently at a site other than the active site
 Alter affinity of E for its S, or modify maximal
catalytic activity of E, or both.
 Can have positive or a negative effectors
 May affect Vmax, Km or both.
 Can be
 homotropic (S is an effector) sigmoidal shape
curve  Cooperativity.
 heterotropic (product or other substance)
feed back inhibition
 Regulation of enzyme by covalent modification
1) Phosphorylation (uses ATP as
phosphate donor) by kinases or
dephosphorylation by
phosphoprotein phosphatases
2) The phosphorylated form of the
enzyme can be more or less
active
 e.g. Upon phosphorylation:
 Glycogen phosphorylase
(glycogen degradation) activity
increases
 Glycogen synthase (glycogen
synthesis) activity decreases
 Induction & repression of enzyme synthesis
• cells can regulate the amount of enzyme present by altering the
rate of enzyme synthesis.
•  (induction) or  (repression) of enzyme synthesis leads to an
alteration in the total population of active sites.
• Alterations in enzyme levels are slow (hours to days), compared
with allosterically regulated changes in enzyme activity, which
occur in seconds to minutes.
Alteration of plasma enzyme levels in disease states
 The activities of many enzymes are routinely
determined for diagnostic purposes in diseases of the
heart, liver, skeletal muscle, and other tissues.

 The level of specific enzyme activity in the plasma

frequently correlates with the extent of tissue damage.

 Determining the degree of elevation of a particular

enzyme activity in the plasma is often useful in
evaluating the prognosis for the patient.
 Plasma enzymes as diagnostic tools
 Some enzymes show relatively high activity in only one
or few tissues.
 The presence of increased levels of these enzymes in
plasma thus reflects damage to the corresponding tissue.
 e.g. alanine aminotransferase (ALT) is abundant in the
liver. The appearance of elevated levels of ALT in plasma
signals possible damage to hepatic tissue.
 Increases in plasma levels of enzymes with a wide tissue
distribution provide a less specific indication of the site of
cellular injury  limits the diagnostic value of many
plasma enzymes.
 lsoenzymes and diseases of the heart
 Isoenzymes (or isozymes) catalyze the same reaction.
 They do not necessarily have the same physical properties because of
genetically determined differences in amino acid sequence.
 isoenzymes may be separated from each other by electrophoresis (different
# of charged of aa)
 Different organs frequently contain characteristic proportions of different
isoenzymes  The pattern of isoenzymes found in the plasma serves as a
means of identifying the site of tissue damage.
 e.g, Plasma levels of creatine kinase (CK) & lactate dehydrogenase (LDH) are
commonly determined in the diagnosis of myocardial infarction.
 They are particularly useful when the electrocardiogram is difficult to
interpret, such as when there have been previous episodes of heart disease.
 Isozymes
• Any of a group of enzymes that catalyze the same reaction
• Have different structures and physical and biochemical properties.
– Lactate dehydrogenase (LDH) - widely distributed through- out
the body
– Cellular damage causes an elevation of the total serum LDH
– Diagnostic usefulness is in the determination of which
fraction of LDH is elevated
• LDH-1 is found mainly in heart muscle and RBC’s
• LDH-2 in white blood cells
• LDH-3 is highest in the lung
• LDH-4 is highest in the kidney, placenta, and pancreas
• LDH-5 is highest in the liver and skeletal muscle
 Diagnosis of myocardial infarction
 Myocardial muscle is the only tissue that contains more than
five percent of the total CK activity as the CK2 (MB) isoenzyme.

 Appearance of this hybrid isoenzyme in plasma (4-8 hr following

onset of chest pain & reaches peak after 24 hr) is virtually
specific for infarction of the myocardium.

 Lactate dehydrogenase activity is also elevated in plasma

following an infarction, peaking 36 to 40 hours after the onset
of symptoms. LDH activity is, thus, of diagnostic value in
patients admitted more than 48 hours after the infarction
 Newest markers for myocardial infarction
 Troponin T and troponin I are regulatory
proteins involved in myocardial
contractility.
 They are released into the plasma in
response to cardiac damage.
 Cardiac troponin I (cTnI) appears in plasma
within 4-6 hr after a myocardial infarction,
peaks in 8-28 hr, & remains for 3-10 days.
 Elevated serum troponins are more
predictive of adverse outcomes in unstable
angina or myocardial infarction than the
conventional assay of CK2