Module 3 - Enzymes

ENZYMES
- are biological catalyst
ENZYME CHARACTERISTICS
 The basic function of an enzyme is to increase the rate of a reaction
 Most enzymes act specifically with only one reactant (called a substrate) to
produce products
 The most remarkable characteristic is that enzymes are regulated from a
state of low activity to high activity and vice versa

Enzyme Structure
 Most enzymes are proteins
 Enzymes may require a non-peptide component as a cofactor. The peptide
component is called the apoenzyme, the cofactor is called as the coenzyme
and the combined functional unit is the holoenzyme
 Cofactors that are tightly bound to the polypeptide are called prosthetic
groups. Such proteins are called as complex or conjugated proteins. Proteins
without prosthetic groups are simple proteins

APOENZYME
 May be inactive in its original synthesized structure
PROENZYME OR ZYMOGEN
 The inactive form of the apoenzyme
 May contain several extra amino acids in the protein which are removed, and
allows the final specific tertiary structure to be formed before it is activated as
an apoenzyme
COFACTOR
 A non-protein substance which may be organic and called coenzyme
 Common coenzymes are vitamins and metal ions
 Another type of cofactor is an inorganic metal ion called a metal ion activator
 Are inorganic and may be bonded through coordinate covalent bonds
 Metal ions as Zn+2, Mg+2, Mn+2, Fe+2, Cu+2, K+1, and Na+1 are used in
enzymes as cofactors

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Vitamins as Coenzymes
Vitamin Coenzyme Function
nicotinamide adenine dinucleotide
niacin oxidation or hydrogen transfer
(NAD+)
riboflavin flavin adenine dinucleotide (FAD) oxidation or hydrogen transfer
pantothenic acid coenzyme A (CoA) Acetyl group carrier
vitamin B-12 coenzyme B-12 Methyl group transfer
thiamin (B-1) thiaminpyrophosphate (TPP) Aldehyde group transfer

PROSTHETIC GROUPS
 Are tightly incorporated into protein structure by covalent or noncovalent forces
 Examples include derivatives of B vitamins such as pyridoxal phosphate, flavin
mononucleotide (FMN), flavin adenine dinucleotide (FAD), thiamin
pyrophosphate, biotin and METAL IONS of Co, Cu, Mg, Mn, and Zn.
 METALLOENZYMES – enzymes that contain tightly bound metal ions

NOMENCLATURE
 The commonly used names for most enzymes describe the type of reaction
catalyzed, followed by the suffix –ase.
 Dehydrogenases – remove hydrogen atoms
 Proteases – hydrolyze proteins
 Isomerases – catalyze rearrangement in configuration
 Modifiers may precede the name to indicate;
(a) the substrate (xanthine oxidase)

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(b) the source of the enzyme (pancreatic ribonuclease)

(c) its regulation (hormone-sensitive lipase)
(d) a feature of its mechanism of action (cysteine protease)
 Alphanumeric designators may be added to identify multiple form s of an
enzyme ( eg., RNA polymerase III; protein kinase C )
 Some enzymes retain their original trivial names, which give no hint of the
associated enzymatic reaction
 Examples are pepsin, trypsin, and chymotrypsin which catalyzes the
hydrolysis of proteins

Classification of Enzymes

Based on catalyzed reactions, the nomenclature committee of the International

Union of Biochemistry and Molecular Biology (IUBMB) recommended the
following classification
1. OXIDOREDUCTASES
 Catalyze a variety of oxidation-reduction reactions
 Common names include dehydrogenase, oxidase, reductase and catalase
2. TRANSFERASES
 Catalyze transfers of groups (acetyl, methyl, phosphate, etc.).
 Common names include acetyltransferase, methylase, protein kinase and
polymerase.
 The first three subclasses play major roles in the regulation of cellular
processes.

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The polymerase is essential for the synthesis of DNA and RNA.

Three major regulatory chemical reactions.

(a) Acetylation - addition of an acetyl group to lysine's R group by

acetyltransferase.
(b) Methylation - addition of a methyl group to DNA's base (e.g. cytosine) by
methylase.
(c) Phosphorylation - addition of a phosphate group to the R group of tyrosine,
serine or threonine (only tyrosine is shown here) by protein kinase.
3. HYDROLASES
Catalyze hydrolysis reactions where a molecule is split into two or more
smaller molecules by the addition of water
 PROTEASES split protein molecules
 HIV protease is essential for HIV replication
 Caspase plays a major role in apoptosis
 NUCLEASES split nucleic acids (DNA and RNA)
 Based on the substrate type, they are divided into RNase and DNase.
 RNase catalyzes the hydrolysis of RNA

 DNase acts on DNA

 They may also be divided into exonuclease and endonuclease.

 The exonuclease progressively splits off single nucleotides from one end

of DNA or RNA.
 The endonuclease splits DNA or RNA at internal sites.

 PHOSPHATASE catalyzes dephosphorylation (removal of phosphate groups).

 Example: calcineurin
 The immunosuppressive drugs FK506 and Cyclosporin A are the
inhibitors of calcineurin
4. LYASES
 Catalyze the cleavage of C-C, C-O, C-S and C-N bonds by means other than
hydrolysis or oxidation.
 Common names include decarboxylase and aldolase.

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5. ISOMERASES
 Catalyze atomic rearrangements within a molecule.
 Examples include rotamase, protein disulfide isomerase (PDI), epimerase and
racemase

* The reactions catalyzed by the two enzymes can assist a peptide chain to fold
into a correct three-dimensional structure

6. LIGASES
 Catalyze the reaction which joins two molecules
 Examples include peptide synthase, aminoacyl-tRNA synthetase, DNA ligase
and RNA ligase

The IUBMB committee also defines subclasses and sub-subclasses

 Each enzyme is assigned an EC (Enzyme Commission) number
 For example, the EC number of catalase is EC1.11.1.6
 The first digit indicates that the enzyme belongs to oxidoreductase (class
1)
 Subsequent digits represent subclasses and sub-subclasses

Mechanism of Enzyme Action

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Mechanism of Enzyme Action

I. Lock and Key Theory

 first postulated in 1894 by Emil Fischer

 The lock is the enzyme and the key is the substrate
 Only the correctly sized key (substrate) fits into the key hole (active site) of the
lock (enzyme)

II. Induced Fit Theory

 Postulated by Daniel Koshland

 It states that, when substrates approach and bind to an enzyme they induce a
conformational change
 This change is analogous to placing a hand (substrate) into a glove (enzyme)
 Assumes that the substrate plays a role in determining the final shape of the
enzyme and that the enzyme is partially flexible.

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 This explains why certain compounds can bind to the enzyme but do not react
because the enzyme has been distorted too much
 Other molecules may be too small to induce the proper alignment and
therefore cannot react
 Only the proper substrate is capable of inducing the proper alignment of the
active site

MECHANISMS TO FACILITATE CATALYSIS

A. CATALYSIS BY PROXIMITY
 For molecules to react, they must come within bond-forming distance of
one another
The higher the concentration, the more frequently they will encounter
one another and the greater will be their rate of interaction
B. ACID-BASE CATALYSIS
 Can be specific or general
“Specific” meaning only protons (H3O+ , specific acid) or OH- ions

(specific base)
C.CATALYSIS BY STRAIN
 Strain is created by binding to substrates in a conformation slightly
unfavorable for the bond to undergo cleavage
The strain stretches or distorts the targeted bond, weakening it and

making it more vulnerable to cleavage
D.COVALENT CATALYSIS
 Involves the formation of a covalent bond between the enzyme and one or
more substrates
 Introduces a new reaction pathway with lower activation energy thus faster
than the reaction pathway in homogenous solution
 Common among enzymes that catalyze group transfer reactions

Part II. ENZYME KINETICS

The field of biochemistry concerned with the quantitative measurement of
the rates of enzyme-catalyzed reactions and the systematic study of factors that
affect these rates
REACTION MODEL

E+S  ES  E + P
where S is the substrate
E is the enzyme
ES is the enzyme-substrate complex
k1, k-1, and k2 are rate constants

MICHAELIS MENTEN EQUATION

 Describes how reaction velocity varies with substrate concentration
Vo = Vmax S
Km + S

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where vo = initial reaction velocity

Vmax = maximal velocity
Km = Michaelis constant (k-1 + k2)/k1
S= substrate concentration

ASSUMPTIONS
 Relative concentrations of E and S
– S >E, so [ES] at any time is small
 Steady-state assumption
– [ES] does not change in time
– E + S = ES = E + P, the rate of formation of ES is equal to that of the
breakdown of ES
 Initial velocity
– Used in the analysis of enzyme reactions
– Rate of reaction is measured as soon as E and S are mixed
– P is very small, the rate of back reaction from P to S can be ignored

CONCLUSIONS
1. Characteristics of Km
a. Small Km
 reflects high affinity of the E for S because a low concentration of S is
needed to half-saturate the enzyme – that is, reach a velocity that is ½ Vmax
b. Large Km
 Reflects low affinity of E for S because a high concentration of S is needed
to half-saturate the enzyme
2. Relationship of velocity to enzyme concentration
 The rate of reaction is directly proportional to the enzyme concentration at all
substrate concentrations
3. Order of reaction
 First order - S < Km, the velocity of reaction is roughly proportional to the
enzyme concentration
 Zero order - S > Km, the velocity is constant and equal to Vmax; the rate of
reaction is then independent of substrate concentration

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Lineweaver-Burk Plot
 Also called a double-reciprocal plot
 If 1/v0 is plotted VS 1/[S], a straight line is obtained
 The intercept on the x-axis is equal to -1/Km
 The intercept on the y-axis is equal to 1/Vmax

 Can be used to calculate Km and Vmax as well as to determine the mechanism

of enzyme inhibitors
 Equation describing the Lineweaver-Burk Plot is:

INHIBITION OF ENZYME ACTIVITY

 INHIBITOR – substance that can diminish the velocity of an enzyme
catalyzed reaction

TYPES OF INHIBITION
 COMPETITIVE INHIBITION
 NONCOMPETITIVE INHIBITION
COMPETITIVE INHIBITION
 Inhibitor binds reversibly to the same site that the substrate would normally
occupy, and therefore competes with the substrate for that site
 Inhibitors tend to resemble the structures of a substrate, and thus are termed
as substrate analogs

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 Malonate (¯OCOCH2COO¯) competes with Succinate

(¯OOCCH2CH2COO¯) for the active site of succinate dehydrogenase
(SDH)
 SDH catalyze the removal of one H atom from each of the 2 methylene C’s of
succinate

Consequences of competitive inhibition

 Vmax is unchanged: At high levels of substrate all of the inhibitor is displaced
by substrate.
 Km is increased: Higher substrate concentrations are required to reach the
maximal velocity.

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NONCOMPETITIVE INHIBITION
 Inhibitor and substrate bind at different sites on the enzyme
 The inhibitor binds to both E and ES
 The noncompetitive inhibitor binds to an allosteric site (different location than
the active site) of an enzyme

 The binding of an inhibitor to the allosteric site alters the shape of the enzyme,
resulting in a distorted active site that does not function properly.

Effect of Enzyme inhibition on Lineweaver-Burk plot

NONCOMPETITIVE INHIBITION
 Vmax is decreased: At high levels of substrate the inhibitor is still bound.
 Km is not changed: Noncompetitive inhibitors do not interfere the binding of
substrate to enzyme

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FACTORS AFFECTING ENZYME REACTIONS

I. SUBSTRATE CONCENTRATION
The rate of enzyme catalyzed reaction increases with substrate concentration
until a maximal velocity (Vmax) is reached

2. Effect of temperature
 The rate of enzyme-catalysed reactions increases as the temperature rises to
the optimum temperature
 Above a certain temperature, activity begins to decline because the enzyme
begins to denature

3. Effect of pH
 Each enzyme has an optimal pH
 In order to interact, the E and S have specific chemical groups in ionized or
unionized state
 Amino group in protonated form (-NH3+)  increase catalytic activity
 At alkaline pH, amino group is deprotonated  decrease in rate of reaction
 Extremes of pH can lead to denaturation

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REGULATION OF ENZYME ACTIVITY

A. ALLOSTERIC REGULATION
B. REGULATION OF ENZYMES BY COVALENT MODIFICATION

A. ALLOSTERIC REGULATION
EFFECTORS – molecules that regulate allosteric enzymes that bind
noncovalently at a site other than the active site
 Negative effectors – inhibit enzyme activity
 Positive effectors – increases enzyme activity

HOMOTROPIC EFFECTORS
 Substrate itself serves as an effector
 Most often a positive effector
 The presence of a substrate molecules at one site on the enzyme enhances
the catalytic properties of the other substrate-binding sites(their sites exhibit
cooperativity)

HETEROTROPIC EFFECTORS
 The effector may be different from the substrate

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Feedback Inhibition

B. REGULATION OF ENZYMES BY COVALENT MODIFICATION

 Most frequently by the addition or removal of phosphate group from specific
Ser, Thr, and Tyr residues of the enzyme
Response of Enzyme to phosphorylation
 Phosphorylated form may be more or less active than the unphosphorylated
enzyme
 Glycogen phosphorylase (degrades glycogen) activity is increased
 Glycogen synthase (synthesize glycogen) activity is decreased
INDUCTION and REPRESSION of enzyme synthesis
 Alter the total population of active sites rather than influencing the efficiency of
existing enzyme molecules
 Enzymes that are needed at only one stage of development or under selected
physiologic conditions are subject to regulation of synthesis
 Enzymes that are in constant use are regulated by altering the rate of enzyme
synthesis

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Mechanisms for Regulating Enzyme Activity

Regulator event Typical effector Results Time required for

change
Substrate Substrate Change in velocity Immediately
Availability
Product inhibition Product Change in Vmax and/or Km Immediately
Allosteric control End product Change in Vmax and/or Km Immediately
Covalent Another enzyme Change in Vmax and/or Km Immediately -
modification minutes
Synthesis or Hormone or Change in the amount of Hours to days
degradation of metabolite enzyme
enzyme

ENZYMES IN CLINICAL USE

1. Enzyme inhibitors as DRUGS
2. Enzymes in CLINICAL DIAGNOSIS

Enzyme inhibitors as DRUGS

 STATINS – HMG Coenzyme A reductase inhibitors; lower serum lipid
concentration
 EMTRICTABINE and TENOFOVIR DISOPROXIL FUMARATE – inhibitors of
viral reverse transcriptase; block replication of HIV
 ACE Inhibitors (Captopril, Lisinopril, Enalapril) – antihypertensive agents
 Lactam Antibiotics (Penicillin and Amoxicillin) – inhibitors of alanyl alanine
carboxypeptidase-transpeptidase, thus blocking cell wall synthesis

Enzymes in CLINICAL DIAGNOSIS

2 GROUPS OF PLASMA ENZYMES
 Actively secreted into the plasma by certain organs
 Released from the cells during normal cell turnover
 Intracellular, have no physiologic function in the plasma
 Constant level in healthy individuals and represent a steady state
* Elevated enzyme activity in the plasma may indicate tissue damage
accompanied by increased release of intracellular enzymes, thus useful as a
diagnostic tool

ISOENZYMES
 Also called isozymes
 Enzymes that catalyze the same reaction but differ in their physical properties
because of genetically determined differences in amino acid sequence
 Different organs frequently contain characteristic proportions of different
isoenzymes
 Isoenzymes found in the plasma serve as a means of identifying the site of
tissue damage

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CK, Creatinine kinase

 – also called Creatinine phosphokinase (CPK)
 3 isoenzymes; CK1, CK2, and CK3
 Each isoenzyme is a dimer composed of 2 polypeptides (B and M subunits:
CK1=BB, CK2=MB, CK3=MM)
 CK2(MB) isoenzyme is present in more than 5% in myocardial muscles
 Appears approximately 4 to 8 hours following onset of chest pain, and reaches
a peak in activity at approximately 24 hours

LACTATE DEHYDROGENASE (LDH)

 Elevated following an infarction peaking 3 to 6 days after the onset of
symptoms
 Of diagnostic value in patients admitted more than 48 hours after the infarction

Principal Serum Enzymes Used in Clinical Diagnosis

Serum Enzyme Major Diagnostic Use

Aminotransferases
 Aspartate aminotransferase  Myocardial infarction
(AST, or SGOT)
 Alanine aminotransferase  Viral hepatitis
(ALT, or SGPT)
Amylase Acute pancreatitis
Ceruplasmin Hepatolenticular degeneration (Wilson’s
disease)
Creatinine kinase Muscle disorders and myocardial infarction

-Glutamyl transpeptidase Various liver diseases

Lactate dehydrogenase Myocardial infarction
(isoenzymes)
Lipase Acute pancreatitis
Phosphatase, acid Metastatic carcinoma of the prostate

Various bone disorders, obstructive liver

Phosphatase, alkaline diseases

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THE ENZYME WARP

Based on "The Time Warp"
Original Words and Music by Richard O'Brien (?)

It’s an enzyme:
One of those globular
Proteins - with a twist:
It does reactions
That you can’t do in test-tubes -
‘Cos it’s a catalyst!!

It finds routes of low activation

Energy for reactions, when
The substrate’s binding
And the complex is forming:
It’s the enzyme warp again!
It’s the enzyme warp again!

It’s a protein catalyst

With high specificity-y-y-y-y!
Induced fit hypothesis
Or lock and key theory!
But it’s the active si-ite
That really mangles that su-u-u-u-ubstrate!
It’s the enzyme warp again!
It’s the enzyme warp again!

There’s ligases
Oxidoreductases
Transferases
Everywhere;
It can do oxidations
Or isomerisations -
If its coenzyme’s there;

Active site competition

Can cause inhibition
Increasing Km;
It can end up denatured
With a high temperature -
It’s the enzyme warp again!
It’s the enzyme warp again!

It’s the enzyme warp again!

It’s the enzyme warp again!

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