Lecture/s Title: ENZYMES AND ITS KINETICS

Subject: BIOCHEMISTRY Module # 1 Lecture # 6

Lecturer: DR. MELLITON BATO Date: 8/29/17
Transcribers: AGAS, ALLAS, ARTETA (Checked by: MOLINA G)

OUTLINE CLINICAL CASE

A. AMI
I. Clinical Case
II. Enzymes
III. Kinetics
IV. Enzyme Kinetics
V. Enzymatic Inhibition
VI. ACE and CK

REFERENCES (CITE ALL REFERENCES!!!)

• Lecture of Dr. Meliton Bato

 Engle M, Linick JM, Pearlstein G, Wilkinson MB (eds.).

2012. MCAT Biology Review, 3/e. New York (NY):
Kaplan Publishing.
 Harvey RA, Ferrier DR. (eds.). 2011. Lippincott’s
Illustrated Reviews: Biochemistry, 5/e. Baltimore (MD):
Lippincott Williams & Wilkins.
 Murray RK, Bender DA, Botham KM, Kennelly PJ,
Rodwell VW, Weil PA. 2012. Harper’s Illustrated
Biochemistry, 29/e. New York (NY): McGraw-Hill.

Legend:

Remember
Lecturer Book
(Exams)
 

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 ENZYMES AND ITS KINETICS

ISOZYMES NON PROTEIN MOLECULES NEEDED BY

ENZYMES
• Genetic variants of an enzyme
• Genetically independent proteins with little homology • Metallic, nonprotein helper: COFACTOR
• Heteropolymers of two or more noncovalently bound • Organic, nonprotein helper: COENZYME
polypeptide chains • Coenzymes that are not covalently binded:
COSUBSTRATES
ENZYMES • Coenzymes that are covalently binded: PROSTHETIC
DEFINITION • GROUPS
 Enzymes are protein catalysts
 Substrate: molecule for which a particular enzyme
catalyzes a reaction
o Enzymes interact very specifically with their
substrates
 Enzymes direct all metabolic events

PROPERTIES SIGNIFICANCE
• Contain active sites
• special pocket/cleft that participate in substrate
binding and catalysis
•  Forms an ES complex
• Binding is a conformational change in the enzyme
• Has catalytic efficiency
• Are highly specific
• Catalyzes only one chemical reaction
• Require helpers

Holoenzyme: active enzyme with its nonprotein

component
Apoenzyme: active enzyme without its nonprotein
component

• Are regulated
• Are compartmentalized

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 ENZYMES AND ITS KINETICS

CLASSES ▪ short recommended name

▪ complete systematic name

• Systematic name:

 Potentially confusing enzyme nomenclature:

▪ Synthetase- requires atp
▪ Synthase – no atp required
▪ Phosphatase- uses water to remove
phosphoryl group
▪ Phosphorylase- uses Pi to break a bond
and generate a phosphorylated product
▪
Dehydrogenase- NAD+/FAD electron
acceptor in redox
▪ Oxidase- O2 receptor, atoms not
incorporated to substrate
▪ Oxygenase- 1 or 2 atoms incorporated

LAW OF MASS ACTION

• Defines how a reaction moves
• In theory, all chemical reactions are reversible
• S ↔ P Increasing molar concentrations of the
substrate will move the reaction forward
• Increasing the product will move the reaction
backward

 Part of Le Chatelier’s Principle also known as the

equilibrium law, which states that a system to which a
“stress” is applied tends to shift as to relieve the applied
stress.

NOMENCLATURE
 Each enzyme is assigned two names.
Transcribed by: AGAS, ALLAS, ARTETA
EQUILIBRIUM CONSTANT
• THE EQUILIBRIUM CONSTANT DESCRIBES THE
EQUILIBRIUM OF THE REACTION
• Keq is the ratio of product concentration to substrate
concentration at equilibrium
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 ENZYMES AND ITS KINETICS

• Entropy change (Δ S ) measure of the randomness or

disorderliness of the system
𝒌𝟏 [𝑪][𝑫] 𝒄𝒐𝒏𝒄𝒆𝒏𝒕𝒓𝒂𝒕𝒊𝒐𝒏 𝒐𝒇 𝒑𝒓𝒐𝒅𝒖𝒄𝒕𝒔
= = = 𝑲𝒆𝒒 • Free energy change (Δ G) the driving force of the
𝒌𝟐 [𝑨][𝑩] 𝒄𝒐𝒏𝒄𝒆𝒏𝒕𝒓𝒂𝒕𝒊𝒐𝒏 𝒐𝒇 𝒓𝒆𝒂𝒄𝒕𝒂𝒏𝒕𝒔
reaction
S + E ↔ P +E Keq= [S][E] / [P][E]
∆𝑮 = 𝑯 − 𝑻∆𝑺
 The equilibrium constant is the ratio of the amount of Second Law of Thermodynamics – Concept of
products over the amount of reactants when equilibrium is Entropy
reached. It describes a set of reactions only when chemical
 The second law of thermodynamics states that energy
equilibrium is occurring.
spontaneously disperses from being localized to becoming
 The value of Keq can be used to determine the direction of
spread out is not hindered from doing so.
the reaction
o If Keq > 1, this means that the rate of forward
 The second law of thermodynamics is not an equation or a
quantitative relationship but rather a statement of
reaction is faster than the rate of reverse
impossibility. It states that thermodynamic processes that
reaction, that is forward reaction is favored
occur in nature are all irreversible processes. These are
o If Keq < 1, this means that the reverse
processes that proceed spontaneously in one direction but
reaction is favored.
not the other. However, the second law can be stated as a
o If Keq = 1, this means that the rate of forward
quantitative relationship with the concept of entropy.
and reverse reaction is the same and that the
net direction of the reaction is neither  Entropy (S) is often described as a measure of how spread
forward nor reverse. out or dispersed the energy of a system is among the
different possible ways that system can contain energy. The
I don’t want to go into the technical details of this but simply put greater the dispersal (or “disorder”) the greater is the
the forward and reverse reactions still occur it’s just that, for entropy. Entropy can be measured according to the
example, if Keq > 1, then 10 forward reactions occur at a given equation:
instant, while 1 reverse occurs. 𝑸
𝜟𝑺 =
Forward AND reverse occur simultaneously it’s just that one is 𝑻
favored over the other depending on the Keq.
The analogy of entropy as to disorder is actually really confusing
Shift right At equilibrium Shift left (and probably erroneous even). According to statistical mechanics,
entropy is a measure of the spontaneous dispersal of energy at a
i.e., toward the i.e., toward the
product, away from reactant, away from specific temperature or how widely spread out energy becomes in a
the reactant the product process. If you take a look at the semantics of the second law, you
would notice that it never ever mentions that it could never be re-
concentrated (again). The second law only states that the
spontaneous change from being dispersed (i.e., “disorder”) to
becoming concentrated is, statistically speaking, highly unlikely to
occur.
• Increasing • Removing [reactant]
[reactant] • Increasing [product] In short, ‘pag tinanong kung gaano kataas ang likelihood na
• Removing [product] • Decreasing the magkabalikan kayo ni ex-… highly unlikely! #HugotPaMore
• Increasing the temperature of the
temperature of the endothermic reaction
endothermic • Increasing the
reaction temperature of the
• Decreasing the exothermic reaction
temperature of the • Increasing pressure
exothermic reaction when the moles of
• Increasing pressure gaseous reactants is
when moles of less than moles of
gaseous reactants gaseous product
is greater than
moles of gaseous
product

FREE ENERGY

• Enthalpy change (Δ H) difference in the total

chemical bond energies between the substrates and
products

∆𝑯 = ∆𝑬 + 𝑷 ∗ ∆𝑽

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 ENZYMES AND ITS KINETICS

reaction (i.e., it requires energy;

endergonic) (Figure 2).

THE FREE ENERGY CHANGE IS THE DRIVING

FORCE FOR CHEMICAL REACTIONS ∆H ∆S ∆G; Outcome
- + ∆G < 0; spontaneous at all
 Gibbs free energy (G), or simply free energy is the temperatures
thermodynamic function used to express the spontaneity of
a reaction. + - ∆G > 0; non-spontaneous at all
temperatures
𝑮 = 𝑯 − 𝑻𝑺
 The change in free energy (∆G) of a system for a constant- + + Spontaneous only at high
temperatures (∆G < 0 when oC ↑)
temperature process is:
∆𝑮 = ∆𝑯 – 𝑻∆𝑺 - - Spontaneous only at low
temperatures (∆G < 0 when oC ↓)
 The value of ∆G tells us whether a reaction happens
spontaneously or not. When ∆G > 0, a reaction is non-
spontaneous and is said to be endothermic. On the other
One of the most common pitfall questions when it comes to
hand, when ∆G < 0, a reaction is spontaneous and is said
thermodynamics is this:
to be exothermic. Note that spontaneous describes as to
whether the reaction will carry out without the input of When ∆ G < 0, does it mean that the reaction proceeds rapidly?
additional energy, it does not necessarily mean it occurs Answer: When ∆G < 0, it means that a reaction is SPONTANEOUS
quickly (requires no additional energy to proceed)… BUT NOT
o When ∆G < 0, then the reaction is RAPID/FAST.
spontaneous and energy is released
Problem solving
(exergonic)
1.In the hypothetical enzymatic reaction A → B, what is the
Keq formula for this reaction?
2. If A’s concentration is 1 while B’s is 20, what is the free
Gibbs energy ?

3. Will the reaction move forward or backward if this

o When ∆G > 0, then the reaction is not
spontaneous and energy is added to the
Transcribed by: AGAS, ALLAS, ARTETA
reaction was in equilibrium?
ANSWERS:
1. The Keq formula is the dividend of the products of the
product and the products of the substrate. Keq = P1
xP2…./S1 xS2…. Keq = [B]/[A]
2. The Keq of the reaction in equilibrium is [B]/[A], therefore
Keq = 20/1 = 20 G0’, use the formula: freeTo compute for
the free Gibbs energy or G0’ = -1.364 x log KeqGibbs
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 ENZYMES AND ITS KINETICS

energy or G0’ = -1.364 x log (20) = -1.364 x1.301 =free

Gibbs energy or -1.77 kcal/mol G0’, rearrange the
formulaTo convert it back to G0’/-1.364) = KeqAntilog (
Antilog function is the 10x in the scientific calculator
3. The reaction in equilibrium will proceed forward

SUBSTRATE BINDING
ENZYMES ARE BOTH POWERFUL AND SELECTIVE

• Almost all enzymes are globular proteins

• Enzymes can accelerate a chemical reaction
• It is defined as the maximal number of substrate
molecules converted to product by one enzyme
molecule per second
• Enzyme-catalyzed reactions are highly efficient,
proceeding from 103–108 times faster than
uncatalyzed reactions

When an enzyme is inhibited by a drug or is deficient because of a KINETICS

genetic defect, only one reaction is blocked
THE SUBSTRATE MUST BIND TO ITS ENZYME
BEFORE THE REACTION CAN PROCEED
• In the enzyme-substrate, complex, the substrate is
bound noncovalently to the active site on the surface
of the enzyme protein
• The active site contains the functional groups
• If a prosthetic group participates in the reaction as a
coenzyme, it is present in the active site
MODELS ON SUBSTRATE BINDING
• The lock-and-key model of substrate binding to the
enzyme active site
• The induced-fit model of substrate binding to the
enzyme active site
• The induced-fit model postulates an initial weak,
flexible interaction of the substrate with groups in the
enzyme’s substrate (ES) binding site.
ACTIVE SITE
• MEDIATES ENZYME ACTIVITY
• relatively small
• three-dimensional structures that are formed as a
result of the overall tertiary structure of the protein
• occur in clefts and crevices in the protein
ENZYMATIC SPECIFICITY ON A SUBSTRATE IS
DEPENDENT ON THE GEOMETRY OF E-S COMPLEX
• The enzyme's substrate specificity is determined by
the geometry of enzyme-substrate binding
• If the substrate is optically active, generally only one
of the isomers is admitted
• A three point attachment is the minimal requirement
for stereoselectivity
CHYMOTRYPSIN

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 ENZYMES AND ITS KINETICS

REACTION RATE
• The rate (velocity) of a chemical reaction can be
described by a rate constant k:

• In this one substrate reaction, the reaction rate is

defined by

• For a reversible reaction, the forward and backward

reactions must be considered separately:

• At equilibrium, V forward = V backward . Therefore the

net reaction is zero

• In a first order reaction, the reaction rate is directly

proportional to the substrate concentration.
• When the substrate concentration [A] is doubled, the
reaction rate V is doubled as well
• Uncatalyzed one-substrate reactions follow first-order
kinetics

A+B→C+D

• A zero-order reaction is independent of the substrate

concentration
• Zero-order kinetics are observed only in catalyzed
reactions

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 ENZYMES AND ITS KINETICS

Factors Affecting Reaction Rate

Substrate concentration

 As the amount of substrate increases, so does the rate of the

reaction

Maximal velocity:

• The rate of an enzyme-catalyzed reaction increases

with substrate concentration until a maximal velocity
(Vmax) is reached
• The leveling off of the reaction rate at high substrate
concentrations reflects the saturation with substrate of
all available binding sites on the enzyme molecules
present.

Hyperbolic shape of the enzyme kinetics curve:

 Michaelis-Menten kinetics and show a sigmoidal curve

that is similar in shape to the oxygen dissociation curve of
haemoglobin.

ENZYME KINETICS
FREE ENERGY ACTIVATION

ENZYMES DECREASE THE FREE ENERGY OF

ACTIVATION

• In reality, however, many of these reactions do not

occur at a perceptible rate
• The reason is that in both catalyzed and uncatalyzed
reactions, the substrate must pass through a
transition state before the product is formed
• The structure of the transition state is intermediate
between substrate and product, but its free energy
Temperature
content is higher
 The optimum temperature for most human enzymes is The basic principles of an enzyme catalyzed
between 35-40C. reaction are the same as any chemical reaction
 Human enzymes start to denature at temperatures above
When a chemical reaction proceeds, the substrate must gain
activation energy to reach a point called the transition state of the
reaction, at which the energy level is maximum

pH

• The concentration of H+ affects reaction velocity in

several ways. the catalytic process usually requires
that the enzyme and substrate have specific
chemical groups in either an ionized or un-ionized
• Effect of pH on enzyme denaturation: Extremes of pH
can also lead to denaturation of the enzyme

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 ENZYMES AND ITS KINETICS

• The free energy difference between substrate and

transition state is called the free energy of activation ( Relationship of velocity to enzyme concentration:
ΔG act )
• It is an energy barrier that must be overcome by the • The rate of the reaction is directly proportional
kinetic energy of the reacting molecules as they collide to the enzyme concentration at all substrate
with each other. concentrations
• ENZYMES DECREASE THE FREE ENERGY OF LINE-WEAVER BURK PLOT
ACTIVATION
 also called double-reciprocal plot
MICHAELIS-MENTEN
• In 1913, discovered by Leonor Michaelis and
Maud Leonora Menten

EADIE-HOFSTEE PLOT
Second linear form of the Michaelis-Menten equation

ENZYMATIC INHIBITION
Types
A.REVERSIBLE

• Competitive
• Noncompetitive
• Uncompetitive

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 ENZYMES AND ITS KINETICS

B.IRREVERSIBLE REVERSIBLE INHIBITION: NONCOMPETITIVE

NONCOMPETITIVE
• Inhibitor combines covalently with the enzyme so that
physical methods are ineffective in separating the two • Inhibitor binds to a site that is different from the
E.g. organophosphate poisoning substrate binding site

 Non-competitive inhibition involves the binding of the

inhibitor on the allosteric site instead of the active site,
which causes a change in enzyme conformation. The
allosteric site is the non-catalytic region of the enzyme that
bind regulators. Because the two molecules do not compete
for the active site, inhibition is considered non-competitive.
Non-competitive inhibition involves phosphorylation of
REVERSIBLE INHIBITION: COMPETITIVE the active site or alteration of the shape of the active site.
This makes the substrate unable to bind to the active site.
This kind of inhibition can be overcome by increasing the
Because of this, even if the amount of substrate were
amount of substrate relative to the inhibitor as the amount
increased, the rate of the reaction would not increase.
of the inhibitor is increased, the rate of the reaction will
When the non-competitive inhibitor leaves the allosteric
decrease more or less proportionately
site, the active site returns to normal and the substrate can
bind to it again.
 Irreversible even by increasing [S]
 Enzyme-inhibitor complex can still bind the substrate but its
efficiency at transforming substrate to product reflected by
vmax is decreased, Km remains the same
 In the Michaelis-Menten plot this shows up as a plot that
does not reach Vmax (Figure 9).
 For Lineweaver-Burk plots, lines for inhibited reaction
intersect on the x-axis with those for uninhibited reaction.
Since Vmax is decreased the y-value is farther away from
• presence of a competitive inhibitor decreases the 0.
rate of the reaction

 As the name suggests, competitive inhibition occurs when

an inhibitor competes with the substrate for the active site.
The inhibitor is a molecule that must resemble the substrate
chemically and physically, in other words a structural
analog of the substrate. An inhibitor, however, must be
different from the substrate in that binding to the enzyme
does not cause a reaction. The strength of competitive
inhibition depends on the ratio between the inhibitor and the
substrate. If there were a high [inhibitor] to [substrate]
REVERSIBLE INHIBITION: UNCOMPETITIVE
ratio, then inhibition would occur. However, if there were a
low [inhibitor] to [substrate] ratio, then little to no • it usually occurs in a system with more than one
inhibition would occur. substrate Uncompetitive inhibitors bind only to the
 Is reversible by increasing [S] enzyme-substrate complex but not to the free
 vmax remains the same but Km is increased since there is enzyme
no change in conformation of the active site • uncompetitive inhibitors work best when the substrate
concentration is high
 In the Michealis-Menten plot, this shows up as a less steep
plot that reaches Vmax at a greater Km value (Figure 1).
 For Lineweaver-Burk plot lines for the inhibited reaction
intersect on the y-axis with those for the uninhibited
reaction. Since Km is increased the x-value is closer to 0
(Figure 2)

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 ENZYMES AND ITS KINETICS

Figure 2: Lineweaver-Burk plot for enzyme inhibition. Uninhibited

enzyme (in red). In competitive inhibition (in green), Km is increased,
which is indicated by an x-value closer to 0 and an intersection of the its
graph (to that of the uninhibited) on the y-axis. In noncompetitive
inhibition (in blue), Vmax is decreased, which is indicated by a y-value
that is farther away from 0 and an intersection of its graph (to that of the
uninhibited) on the x-axis.

Competitive Non-competitive

Form Substrate analog Doesn’t resemble

substrate analog

Binds to Active site Other sites (allosteric

site)

Inhibits by Binding to the active Changing conformation

site, which prevents of the enzyme or the
the formation of ES- shape of the active site,
complex which affects catalysis

Vmax NO CHANGE DECREASES

Km INCREASES NO CHANGE
Reversible Inhibition - Summary Reversible by YES! NO!
increasing [S]?

Michaelis- Less steep plot A plot that never reaches

Menten compared to vmax
uninhibited

Lineweaver- Intersects at y-axis; Intersects at the x-axis; y-

Burk x-value is closer to 0 value is farther away from
0

Figure 1: Michaelis-Menten plot for enzyme inhibition. Uninhibited

enzyme (in red). In competitive inhibition (in green), the slope of the plot
is less steep (more [S] is needed to reach Km). In noncompetitive
inhibition (in blue), the plot does not reach Vmax.

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 ENZYMES AND ITS KINETICS

Renin Angiotensin Aldosterone System (RAAS) B. Regulation of enzymes by covalent modification

• Protein phosphorylation is recognized as one

of the primary ways in which cellular
processes are regulated
1. Phosphorylation and dephosphorylation:
catalyzed by a family of enzymes called
protein kinases that use adenosine
triphosphate (ATP)
2. Response of enzyme to phosphorylation:
Depending on the specific enzyme, the
phosphorylated form may be more or less
active than the unphosphorylated enzyme
C. Induction and repression of enzyme synthesis

• Cells can also regulate the amount of enzyme

present by altering the rate of enzyme
degradation or, more typically, the rate of enzyme
synthesis
• The increase (induction) or decrease (repression)
of enzyme synthesis leads to an alteration in the
CONTROL OF ENZYME ACTIVITY
total population of active sites
• The regulation of the reaction velocity of enzymes is • Alterations in enzyme levels as a result of
essential if an organism induction or repression of protein synthesis are
• An increase in substrate concentration prompts an slow (hours to days), compared with allosterically
increase in reaction rate or covalently regulated changes in enzyme
activity, which occur in seconds to minutes
A. Regulation of allosteric enzymes
ACE AND CK
• Allosteric enzymes are regulated by molecules called
ACE
effectors (also called modifiers) that bind
noncovalently at a site other than the active site
• The presence of an allosteric effector can alter the
affinity of the enzyme for its substrate
• Effectors that inhibit enzyme activity are termed
negative effectors, whereas those that increase
enzyme activity are called positive effectors

1. Homotropic effectors:
When the substrate itself serves as an
effector, the effect is said to be homotropic
• Most often, an allosteric substrate
functions as a positive effector
•
2. Heterotropic effectors:
The effector may be different from the
substrate, in which case the effect is said to
be heterotropic

End-product feedback inhibition • Aldosterone causes sodium retention in the

collecting ducts of the kidneys resulting in water
conservation and Increase vascular volume
• Cell-bound ACE is called tissue ACE (>90%) and
circulating ACE
• 2 catalytic sites: N-terminal and C-terminal on the
extracellular domain C-terminal catalytic site:
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 ENZYMES AND ITS KINETICS

predominant angiotensin 1 hydrolytic activity N –

terminal catalytic site: bradykinin
• Found predominantly in the endothelial cell
membranes
• Lungs and testes are highly-rich Somatic ACE
(sACE) and testicular ACE (tACE) A single gene
encodes both isoforms utilizing 2 alternative
promoter sites

ACE INHIBITORS

• Nonapeptide TEPROTIDE derived from Bothrops

jararaca
• First effective therapeutic ACE inhibitor but could not
be orally given due to hydrolysis in stomach and
the gut
• A critically positively charged Arg residue in the active
site plus a positively charged Zn ion
• This resulted in 3-mercapto-2-methylpropanoyl-L-
proline (captopril) Skin rash and loss of taste Phe-
containing tripeptides with carboxylate groups
(enalapril and lisinopril) Bradykinin hydrolytic activity
at the N-terminal is not inhibited Vasodilation,
angioedema, hypotension
Creatine Kinase (CK)

• Cytosolic CK and intramitochondrial CK Cytosolic CK:

Isoenzymes M and B MM (skeletal), MB (cardiac) BB
(brain and intestine) Intramitochondrial CK:
nonmuscle or ubiquitous MtCK and sarcomeric
muscle CK
• MtCK: supplier of phosphocreatine that shuttles to the
cytosol and is used as an energy source serving as a
substrate for cytosolic ATPases
• CK-MB is serodiagnostic for MI
• CK: biomarker for skeletal muscle injury
• Neuroleptic malignant syndrome: rare complication
with phenothiazines or other psychotropic medications
causing acute muscle injury
• Muscle rigidity, fever and elevated WBC counts CK-
MB/ CK total maybe elevated
• Serum mtCK: reliable independent predictor of
hepatocellular carcinoma development in pts with
cirrhosis or hepatitis C 9 IU/L Correlates to liver
fibrosis and hepatocellular damage
Happy Aral, let’s do this Batch 2021 😊
-G.M.

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