BIOCHEMISTRY 09/03/2019

Enzymes Shifting 01

Trans 03
Dr. Danilo Menorca
[SUBJECT]

OUTLINE MM/DD/YYYY B. CLASSIFICATION OF ENZYMES

Shifting #
I. Enzyme IV. Mechanisms of Enzyme TransNomenclature
#
A. Definition of Terms Catalysis • System is based on the function rather than structure.
[SUBJECT]
B. Classification of Enzymes A. Acid-Base Catalysis • Suffix: “-ase”
II. Enzyme Catalysis Models Substrate Strain → Changed to indicate:
A. Lock and Key Model B. Covalent Catalysis Substrate e.g. xanthine oxidase
B. Induced-Fit Model C. Transition State Enzyme source e.g. pancreatic ribonuclease
Stabilization regulation, hormone-sensitive lipase
[SUBJECT]
III. Enzyme Kinetics
D. Entropy Effect Mode of regulation e.g. hormone-sensitive
A. Reaction Model
B. Michaelis-Menten Equation E. Ground State lipase
Destabilization Mechanism of action e.g. cysteine protease
C. Lineweaver-Burke Plot
D. Cooperate Binding F. Environmental Parameters
V. Additional Enzyme Important Aspects of Enzyme Naming
E. Inhibition of Enzyme
Activity Characteristics
A. Enzyme Activation • The suffix –ase identifies a substance as an enzyme
F. Enzyme-catalyzed
Reaction with 2+
B. Enzyme Specificity • The suffix –in is still found in the names of some first
VI. Factors Affecting Enzyme enzymes (ex. Pepsin, trypsin.)
Substrates
A. Pathway Control • The type of reaction catalysed by an enzyme is often noted
G. Allosteric Control of with a prefix (ex. Oxidase– enzyme that catalyzes an
B. More on Regulation of
Enzyme Activity oxidation reaction.)
Enzymatic Activity
VII. Clinical Applications • Identity of substrate is often noted in addition to the type of
reaction (ex. Glucose oxidase, pyruvate carboxylase)
• Infrequently, if the type of reaction is not given, the reaction
I. ENZYME involved is hydrolysis.
Table 1. Six Major Classes of Enzymes
Enzymes are efficient catalysts whose stringent specificity SIX MAJOR CLASSES OF ENZYMES
extends to the kind of reaction catalyzed, and typically to a single Class Subclass
substrate. Dehydrogenases
Hydroxylases • Catalyze oxidation
and reduction
A. DEFINITION OF TERMS Class 1: Oxygenases
reactions
Oxireductases Reductases
• Substrate is H+ or an
Catalases
• Apoenzyme - the protein part of the enzyme without any electron donor
Oxidases
cofactors or prosthetic group that may be required for the
Kinases & • Catalyze transfer of
enzyme to be functional.
Phosphomutases functional groups or
• Isozyme - enzymes that differ in amino acid sequence Transaldolase & moiety (methyl,
but catalyze the same chemical reaction Transketolase phosphoryl or
• Substrate Binding Site - particular region on the glucosyl) from a donor
enzyme surface where specificity resides Class 2: to an acceptor
• Enzyme Active Site - place at which catalysis occurs Transferases Acyl, Methyl, • Donor is usually a
• Enzyme Allosteric Site - a site in the enzyme where a Glucosyl and coenzyme carrying
molecule that is not a substrate may bind, can either increase Phosphoryl group
or decrease enzyme activity. Transferases
• Rate of chemical reaction - the quantity of a reactant • Reaction type:
𝐴𝐵+𝐶↔𝐴+𝐵𝐶
consumed or the quantity of a product formed in unit time
Phospholipases • Catalyze hydrolysis
• Velocity of a chemical reaction - the change of molar
Ribonucleases of various bonds
concentration of one of reacting substances in a given period of • Cleavage of chemical
Phosphatases
time bond by transfer of
Glycosidases
• Holoenzyme - active compound produced by the combination Class 3:
Deaminases water
of an enzyme with a cofactor Hydrolases
Peptidases
• Cofactors - small organic or inorganic molecules that an Esterases • Reaction type:
apoenzyme requires for its activity Thiolases 𝐴𝐵+𝐶↔𝐴𝑂𝐻+𝐵𝐻
- can associate either directly with the enzyme or in the form of a Amidases
cofactor-substrate complex Decarboxylases • Catalyze the
• Prosthetic groups - cofactors that are tightly and stably Dehydratases cleavage of C-C, C-O,
incorporated into the enzyme’s structure by covalent or Hydratases and C-N bonds by
noncovalent bonds Aldolases means other than
Class 4:
● Substrate – the molecule acted upon by the enzyme to form the Synthases hydrolysis or oxidation
Lyases
product. Since most reactions are reversible, the products of the (non-hydrolytic
forward reaction become substrates for the reverse reaction. cleavage)
Lyases • Reaction type:
𝐴𝐵+𝐶↔𝐴𝑂𝐻+𝐵𝐻
BCHM Concha, Durana, Nicolas, Santos 1 of 9
 Isomerases • Catalyze → Reciprocal changes also occur in the substrate: energy of
Epimerases intermolecular binding is harnessed to facilitate its transformation into
Racemases rearrangements within products
a molecule. Either → As the substrate fits itself and somewhat alters the active
Class 5:
geometric or structural site, it will also alter the substrate itself, which will also
Isomerases
changes. facilitate catalysis.
Mutases
• Reaction type:
III. ENZYME KINETICS
𝐴𝐵𝐶↔𝐴𝐶𝐵
Carboxylases • Catalyze the joining • Study of rates and factors that may cause alterations in rates of
of 2 molecules coupled enzyme-catalyzed reactions
with the hydrolysis of a
pyrophosphate bond Enzymes as catalysts
in ATP or similar • Increase the rate of reaction, but do not affect reaction equilibria
Class 6:
triphosphate forming
Ligases Synthetases C—O, C—S, C—N,
Catalyzed and uncatalyzed reactions
and C—C • Ground state – starting point for either the forward or the reverse
reaction
• Reaction type: • Transition state – top of the energy hill at which decay to the
𝐴𝐵+𝐶↔𝐴𝑂𝐻+𝐵𝐻 substrate (S) and product (P) state is equally probable
• Activation energy (∆Guncat/cat) – difference between the energy
II. ENZYME CATALYSIS MODELS levels of the ground state and the transition state
→ Energy required to surmount a given energy barrier
A. LOCK AND KEY MODEL → Higher activation energy corresponds to a slower
reaction
Emil Fischer • Enzymes lower activation energy (∆Gcat) → faster rate of reaction

Figure 1. Lock and Key Model

• The active site of the free enzyme is complementary in

shape to the substrate.
Figure 3. Effect of enzyme catalysis on activation energy (Leningher, 2008)
• Accounts for enzyme specificity but too rigid, but
molecules in the body are dynamic; dynamic changes A. REACTION MODEL
occur during catalysis.
• Only substrates with the matching shape can fit

B. INDUCED FIT MODEL

Daniel Koshland
• E - enzyme
S - substrate
ES – enzyme-substrate complex
P - product
K1, k-1, k2 – rate constants
• Shows the usual fate of enzyme-catalyzed reactions
Initial Velocity (Vi)
• Change in reactant or product concentration during the first few
seconds of the reaction
• Defines the rate of reaction best since the rate of reaction
decreases over time due to:
→ Decrease in substrate concentration [S]
→ Change in pH
Figure 2. Induced-fit Model → Inhibition of enzyme by the product
→ Denaturation/deactivation of enzyme
• The active site of the enzyme changes shape to • Measured when [S] is much greater than enzyme concentration
accommodate the substrate which improves catalysis. [E]
• Active site is flexible, not rigid.

BCHM Enzymes 2 of 9
Order of reactions B. MICHAELIS-MENTEN EQUATION
• Indicates the rate-limiting step
• Consider the conversion of reactant A to product P 𝑉𝑚𝑎𝑥 [𝑆]
A→P 𝑉𝑖 =
𝑉𝑚𝑎𝑥 + [𝑆]
RATE EQUATION: Vi; [A]
• Quantitative relationship between Vi, Vmax, and [S]
-d[A] • Vmax and Km are constant attributes of an enzyme (not affected
by [E]
------------ = Vi = k[A]n • Michaelis constant (Km) – equivalent to the substrate
concentration at which Vi is half of Vmax
dt → High Km = low binding affinity of the enzyme for substrate
→ Low Km = high binding affinity of the enzyme for substrate
READS: The initial velocity (Vi) depends on the starting
concentration of A ([A]), raised to the nth power, multiplied by a
proportionality constant (k) or RATE CONSTANT. The exponent n
is usually an integer from 1 to 3.

1. FIRST ORDER: A ----------> P vi = k1[A]

2. SECOND ORDER: A + B --> P vi = k2[A][B]

3. THIRD ORDER: 2A + B ---> P vi = k3[A]2[B]

A + 2B ---> P vi = k3[A][B]2

4. ZERO ORDER: Aexcess------> P vi = k0

5. PSEUDO FIRST ORDER:

A + H2O --> P vi = K1[A]

Figure 5. Michaelis-Menten Plot

C. LINEWEAVER-BURK PLOT
• Also called as the double-reciprocal plot
• Linear plot
• Allows more accurate determination of Vmax and Km
• Lineweaver-Burk equation:

Figure 4. Summary of Kinetics of Reactions

Assumptions → Transformed form of Michaelis-Menten Equation
• Relative concentration of E and S → Slope: Km/Vmax; Y-intercept: 1/Vmax; X-intercept: -1/Km
→ [S] >>>> [E]
• Steady state assumption
→ Enzyme-substrate concentration [ES] depends on the amount
of free enzyme and substrate
→ [ES] remains constant over time
→ Velocity of ES complex formation = velocity of its
breakdown

→ Rearranging the terms:

▪ Michaelis constant (Km) = (k2 + k-1)/k1

− Reflects the affinity of the enzyme to the substrate Figure 6. Lineweaver-Burk Plot (1/Vo versus 1/[S])
− Approximates an enzyme’s binding affinity or how well
enzyme binds the substrate D. COOPERATE BINDING
• Some enzymes bind more than one substrate/contain more
than 1 active site (Multimeric enzymes)
• Substrate binding becomes faster as binding sites become
occupied
BCHM Enzymes 3 of 9
 → Positive cooperativity: initial binding increases affinity of
second binding
→ Negative cooperativity: enzymes with single binding site
• At low substrate concentrations: small increase in reaction rate
with increase in [S]
• As [S] further increases, small increase in [S] leads to large
increase in reaction rate
• At very high [S], rate exhibits saturation
• Sigmoidal plot

Figure 9. Competitive inhibition

Figure 7. Sigmoidal saturation kinetics

Hill Equation
• Provides a way to quantify binding by describing the fraction of
saturated ligand binding sites as a function of ligand
concentration

𝑉𝑖
𝑙𝑜𝑔 = 𝑛 log[𝑆] − log 𝑘′
𝑉𝑚𝑎𝑥 − 𝑉𝑖
• n = Hill coefficient
2.Uncompetitive
→ n < 1; no cooperativity
→ Binds at a site distinct from the substrate active site
→ n ≥ 1; cooperativity
→ Binds only to the ES complex
→ Lower Km, lower Vmax

Figure 8. Hill Equation graph

E. INHIBITION OF ENZYME ACTIVITY

• Enzyme inhibitors – molecular agents that interfere with
catalysis, slowing or halting enzymatic reactions
Reversible inhibitors
• Non-covalent bonds = easily removed Fig 10. Uncompetitive Inhibition
1. Competitive
→ Competes with the substrate for the active site of an enzyme
→ Resembles the substrate
→ Reversible by increasing substrate concentration [S]
→ Higher Km, same Vmax

BCHM Enzymes 4 of 9
 Table 2. Summary of reversible inhibitors

INHIBITOR BINDING VMAX KM

Active site of
Competitive Unchanged Increased
the enzyme

Uncompetitive ES complex Decreased Decreased

Non- E or ES
Decreased Unchanged
competitive complex

Irreversible inhibitors
• Bind covalently with the enzyme
• Destroy a functional group on an enzyme that is essential for the
enzyme’s activity
Figure 11. Uncompetitive inhibition
• Useful as affinity labels because it labels the residues at active sites
3. Non-competitive of an enzyme allowing it identification
→ Binds to either E or ES
→ No structural resemblance to substrate F. ENZYME-CATALYZED REACTIONS WITH 2+ SUBSTRATES
→ Same Km, lower Vmax
Sequential Bi-Bi
• Both substrates need to bind
• Single displacement

1. Random
→ Binding can be not in order

2. Ordered
→ Certain substrate needs to bind first
→ Obligatory order for the addition of substrates and release of
products

Ping-Pong Bi-Bi
• 1 substrate, 1 product at a time
• Product is released before all the substrates are bound
• Temporary covalent catalysis
• Double displacement
Figure 12. Non-competitive inhibition

BCHM Enzymes 5 of 9
 G. ALLOSTERIC CONTROL OF ENZYME ACTIVITY B. SUBSTRATE STRAIN
Allosteric modulators/effectors
• Enzymes that catalyze reactions bind substrates on a
1. (+) Modulator conformation slightly unfavorable for the bond that will
→ Requires lower substrate concentration to achieve ½Vmax undergo cleavage
→ Results in hyperbolic plot • Resulting strain distorts the targeted bond to weaken
2. (-) Modulator substrate and cause the substrate to undergo cleavage
→ Requires higher substrate concentration to achieve ½Vmax • Ex. Hydrolytic enzymes
→ Results in sigmoidal plot
Classes of allosteric enzymes
1. K-class
→ Effector alters Km, not the Vmax
2. V-class
→ Effector alters Vmax, not the Km
• Enzymes accelerate the reaction by lowering the
activation energy (Ea)
• Active site of enzyme exhibit high specificity for enzyme-
substrate reactions
• Catalysis by proximity- high substrates, high enzymes → very
close to one another → higher chance to react with one
another
• Enzyme does not change equilibrium constant of reaction; it
only allows equilibrium to be attained more quickly by
lowering activation energy Figure 14. Substrate strain mechanism.
• It can enhance the rates of reaction by a factor of 109 – 1012
times that of a non-catalyzed reaction C. COVALENT CATALYSIS
• Common in lytic reactions (breaking of covalent bonds)
IV. MECHANISMS OF ENZYME CATALYSIS • Formation of a transient covalent bond between the enzyme
and substrate due to attack of nucleophilic or electrophilic
group in the enzyme active site
A. ACID-BASE CATALYSIS • Enzymes become part of the reaction initially but will restore
• Acid- proton donor, base- proton acceptor to original state after the reaction
• The ionizable function groups of the aminoacyl side chains • Introduces a new reaction pathway where activation
and of prosthestic groups contribute to catalysis by acting as energy is lower and therefore faster
acids or bases • Common among enzymes that catalyze group
• Can either be specific or general transfer reactions, residues on enzymes are
→ Specific Acid-Base Catalysis cysteine or serine, occasionally histidine
▪ Rate of reaction is sensitive to changes in • Ping-pong mechanism is exhibited
the concentration of protons but → First substrate is bound and its product released prior to the
independent of the concentration of other binding of the second substrate
acids or bases present in solution or at the
active site
▪ Specific acids are H+ or H2O
▪ Specific base is OH-
→ General Acid-Base Catalysis
▪ Reaction rates are responsive to all the acids or
bases present
▪ Proton transfers mediated by other
classes of molecule
▪ Occurs in vast majority of enzymes Figure 15. Covalent catalysis
▪ General acid/bases are weakly ionizable compounds D. TRANSITION STATE STABILIZATION
• Active state binds the transition state (TS) with a much
greater affinity than the substrate
• Mass action: all substrate converted rapidly to products
• Any factor that increases the population of substrate
molecules resembling the TS will contribute to catalysis

E. ENTROPY EFFECT
• Entropy (S) is the extent of disorder in a system
• At equilibrium, entropy is maximal (2nd Law of
Thermodynamics)
• Decrease in entropy contributes to the rate of reaction by a
factor of 103
• An enzyme with high affinity binding sites for substrates will
cause the substrates to bind with the enzyme in the correct
Figure 13. The active sites of some enzymes contain amino acid functional groups
orientation increasing the effective concentration of
(usually R-Group Functional Groups) that participates in the catalytic process as
proton donors or proton acceptors. reactants, thus, an increase in the reaction rate will occur.
→ PROXIMITY EFFECT
• Decreases entropy
BCHM Enzymes 6 of 9
 • Increases orderliness associated to the proper
orientation of the substrate
• Creates region of high local concentration of substrates
with ideal orientation by the enzyme-substrate complex
Figure 16. Proximity effect (Entropy Effect)
F. GROUND-STATE DESTABILIZATION
● The ground state energy is increased to lower the energy
barrier Ea
● Done by mimicking the TS of the substrate or the
substrate is arranged in its TS
V. ADDITIONAL ENZYME CHARACTERISITICS
A. ENZYME ACTIVATION
• Most enzymes need to become activated first to become useful
• To activate enzymes, there is usually rearrangement of enzyme
molecules
• Ex. most digestive enzymes (stomach and pancreas) are first
produced as inactive precursors:
• ZYMOGENS or PROENZYMES
→ Purpose: protective; to avoid self-destruction and
autodigestion from being active initially
→ Activation = limited proteolysis to realign amino acid
side chains to form the active site and bring crucial amino acids
closer to each other
• Irreversible modification because reunification of the 2 portions
of proteins produced by hydrolysis of a peptide bond is
entropically disfavored.
• Enzymes needed intermittently but rapidly are often secreted in
an initially inactive form since new synthesis and secretions might
not meet the rapid demand.
• Examples: (usually with a prefix “pro-“)
- hormone insulin (proenzyme = proinsulin)
- digestive enzymes: pepsin (pepsinogen)
- trypsin (trypsinogen)
- chymotrypsin (chymotrypsinogen)
- several factors of blood clotting and complement cascades &
connective tissue protein collagen (procollagen)
• Activation of TRYPSINOGEN (229 residues) = removal of N
terminal hexapeptide Val-Asp-Asp-Asp-Asp-Lys (6AA) by
enterokinase to become Trypsin (active) = Conformational
change wherein Ser 183 and His 29 are brought into proximity
• PEPSINOGEN (362 residues) in the stomach is activated by H+
ions in gastric juice → conversion into Pepsin (active) for protein
digestion through the loss of 42 amino acids from N terminal of
the zymogen
CONTROL OF ENZYME CONCENTRATION
BCHM Enzymes
Figure 17. 1.) Formation of a cleavage between AA residue 15
and 16, 2.) Removal of AA residue 14, leaving residues 1-13,
3.) Cleavage between 146 and 149, subunits are then formed,
4.) Disulfide bonds stabilize subunits formed
B. ENZYME SPECIFICITY
• Relative Specificity
→ Enzymes act on the covalent bond in closely related/ same
group of substrates
→ Examples:
▪ Proteolytic enzymes hydrolyze peptide bonds
of polypeptides
▪ Glucosidases act on glycosidic bonds of carbohydrates
▪ Esterases like lipases split ester bonds of lipids
• Absolute Specificity
→ Enzymes act on only one substrate
→ Examples:
▪ Urease acts only on urea
▪ Carbonic Anhydrase acts only on Carbonic Acid
• Stereospecificity
→ Catalyze reactions of only one isomer of a given compound
→ Examples:
▪ Arginase acts only on L-arginine
▪ Glucose Oxidase acts only on β-D-glucose anomer not on
the α-D anomer
• Specificity of proteolytic enzymes:
→ Endopeptidases
▪ bonds attack internal peptide bonds
→Exopeptidases
VI. FACTORS AFFECTING ENZYME
A. PATHWAY CONTROL
• Modulation of activity of one or more key enzymes in a pathway
1. Rate-limiting Enzyme
→enzyme with the lowest Vmax in the pathway
2. Enzyme associated with the committed step
→ enzyme catalyzing the first irreversible reaction in the pathway
• Short-term regulation: regulation occurs via activity modification
of the existing enzyme without change in concentration
• Long-term regulation: refers to changes in amount of enzyme
without altering its kinetic properties
• Cross-regulation: the product of pathway1 is an inhibitor or
activator of an enzyme in pathway
B. MORE ON REGULATION OF ENZYME ACTIVITY
• SYNTHESIS
→ REPRESSORS – by excess metabolites
→ STIMULATORS – by hormones and extracellular signals
→ INDUCERS – for inducible enzymes
7 of 9
 • Cyt p50 ▪ Hexokinase – active at all times for steady supply
• HMG CoA reductase of glucose in the brain
• Enzymes in the Urea Cycle VII. CLINICAL APPLICATIONS
• DEGRADATION
→ Ubiquitin in the Proteasome Pathway
PLASMA ENZYME
→ Covalent Modification
→ Binding with allosteric effector ▪ high concentration in the plasma, is specific to plasma, and
has a functional role in the plasma
→ Association with membrane oligonucleotides or other
proteins
▪ at very low levels in plasma, plays no functional role in the
plasma
CONTROL OF CATALYTIC EFFICIENCY
MEASURING PLASMA ENZYME ACTIVITY
• Directly controlled through change in conformation
▪ NONPLASMA – specific enzymes
• Modulated by activators, inhibitors, or via covalent alterations ▪ PREMISE – changes in the plasma activity; changes that
occurred in the damaged or diseased organ
A. ALLOSTERIC REGULATION
1. Feedback inhibition through a negative allosteric
effector
▪ Kinetics may be competitive, non-competitive, or
mixed
▪ Typically inhibits the first committed step
2. Multiple Feedback Loops
▪ Effect may be strictly additive
▪ Effect is greater than the individual feedback
3. Release of Allosteric 2nd messenger
▪ 1st messenger – hormone or nerve impulse
▪ 2nd messenger – cAMP

COVALENT MODIFICATION
• Irreversible
▪ partial proteolysis
▪ for enzyme activation
▪ enzymes for blood clotting Figure 18. Kinetics of Release of Cardiac Enzymes into Serum
• Reversible After Myocardial Infarction
▪ binding of dissociable ligands
▪ phosphorylation – catalyzed by kinases MEASURING OF NONPLASMA-SPECIFIC
▪ dephosphorylation – hydrolysis of phosphodiester ENZYMES/ISOZYMES IS USED DIAGNOSTICALLY
bond by phosphatase 1. CREATINE KINASE
▪ most common • MB CPK – Acute M.I.
▪ alters the following: • MM CPK – Skeletal muscles
→ enzyme location within cell • BB CPK – Brain tissue
→ enzyme susceptibility to degradation 2. ALKALINE PHOSPHATASE
→ enzyme catalytic efficiency or inefficiency ● in Obstructive Jaundice (CBD stone; Cirrhosis,
→ enzyme responsiveness to allosteric regulation Cancer)
3. LACTATE DEHYDROGENASE
COMPARTMENTALIZATION • LDH1 (H4) – Acute M.I.
• Specific Subcellular Compartment • LDH2 (H3M) – Acute M.I.
▪ In lysosomes – degradation of proteins and • LDH3 (H2M2) – Brain or kidney tissue injury
polysaccharides • LDH4 (HM3) – Skeletal muscle injury
▪ In cytosol – enzymes for fatty acid synthesis • LDH5 (M4) – Liver cell injury (acute hepatitis)
▪ In mitochondria – enzymes for fatty acid oxidation
• Thermodynamic Substitution
• Ability of the enzyme to discriminate between similar
coenzymes
o eg. NAD (catabolism) and NADP (anabolism)

PRESENCE OF ISOZYMES
• these are physically distinct versions of a given enzyme and
they catalyze the same reaction
• mainly products of gene duplication
• confer tissue specificity and adapt tissues to special
circumstance
• provide a backup mechanism
• Example:
→Creatine kinase
▪ allows tissue specificity Figure 19. Enzymes (bold) utilized as reagents
▪ MM – muscle, BB – brain, MB – heart
→Glucokinase (liver) vs. Hexokinase (brain and muscle)
▪ allows the tissue to adapt to special circumstances
▪ Glucokinase – active when there is high blood
sugar
BCHM Enzymes 8 of 9
 Figure 20. ELISA

ENZYMES AS THERAPEUTIC AGENT

STREPTOKINASE
- useful in clearing blood clot that occur in M.I. and lower E.
PLASMIN
- serine protease which cleaves the insoluble fibrin in blood clots
into several soluble components
ASPARAGINASE
- used in treatment of some adult-type leukemia

VIII. REFERENCES
Harper’s Illustrated Biochemistry
Dr. Menorca’s PPT and lecture notes
2020A & 2022 A B C Trans

BCHM Enzymes 9 of 9