Enzymes Biochemistry by Dr. Mudassar Ali Roomi

8/2/2019 Enzymes Biochemistry by Dr. Mudassar Ali Roomi

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ENZYMES

BY

DR. MUDASSAR ALI ROOMI (MBBS, M. Phil)


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ENZYMES

Introduction to enzymes...

Definition of enzymes (biological catalyst).

Biochemical nature of enzymes (usually theseare proteins but may be RNA in nature called

as ribozymes).


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PROPERTIES OF ENZYMES

Enzymes are biologicalcatalysts that increase the velocity ofreaction by lowering the energy of activation.

Enzymes have active sites and may have allosteric sites.

Catalytic efficiency.

Specificity for their substrate (relative or absolute specificity). Enzymes usually need a non protein part for their normal

activity. Holoenzymes = apoenzymes + non protein part.

enzyme activity may be regulated in our body according to

need. Enzymes are located within the cell in compartments.


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NOMENCLATURE OF ENZYMES

Recommended names:these are simple names

which are easy to remember and are commonly used

in practice. These names usually end at the suffix

ase attached to the substrate of the reaction e.g.

lipase, sucrase, uricase and urease etc. some

enzymes retain their original names which do not

give any hint of the associated enzyme reaction e.g.

trypsin and pepsin.

Systematic names:this naming is difficult to

remember and is according to the IUBMB

nomenclature of enzymes (e.g. glyceraldehyde 3-

posphate NAD oxidodeructase).


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Classificationofenzymesaccordingto

IUBMB

Oxidoreductases:These enzymes catalyse the oxidation reduction

reactions e.g. lactic dehydrogenase (LDH)and catalase enzymes.

Transferases:These enzymes catalyze the exchange of groups between

two compounds e.g. aminotransferases (AST and ALT).

Hydrolasesor hydrolytic enzymes:These enzymes catalyze hydrolysis i.e.

decomposition of a compound and break it into two compoundsby

addition of water into the substrate e.g. amylases, lipases, proteinases,

sucrase, maltase, lactase.

Lyases: These enzymes catalyze the removal of a group from a compound

without the addition of water e.g. pyruvate decarboxylase.

Ligases: These enzymes catalyze the reaction of joining two compounds byforming C=O, C-N, C-C, and C-S bonds e.g. pyruvate carboxylase.

Isomerases: These enzymes catalyze the formation of isomers of the

substrate e.g. phosphohexose isomerase.


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DEFINE THE FOLLOWING TERMS

Holo-enzyme = Apoenzyme + non-protein part.

Co-factor .

Co-enzyme (Co-substrate or Prosthetic group).

Lysozymes. Zymogens or pre-enzymes (e.g. pepsinogen,

chymotrypsinogen).


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ISOENZYMES ORISOZYMES.

Definition: these are the enzymes having the same

chemical properties but are different in their

structure.

Isoenzymes ofLDH: LDH1, LDH2, LDH3, LDH4, LDH5.LDH1 is present in heart only and LDH5 is present in

skeletal muscle only while the other isoenzymes are

present both in heart and skeletal muscle.

Isoenzymes ofCPK:CPK-MB (in cardiac muscle), CPK-

BB (in brain tissue), CPK-MM (in skeletal muscle).


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CO-FACTORS


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CO-ENZYMES


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HOW ENZYMES WORK

Template model or lock

and key model.

Induced fit model.


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ENERGY CHENGES OCCURING DURING THE

ENZYME CATALYZES REACTION


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FACTORS AFFECTING REACTION

VELOCITY

1. Effect of Substrate concentration.

2. Effect of Temperature.

3. Effect of temperature pH.4. Effect of enzyme concentration.

5. Effect of product concentration.

6. Effect of activators and coenzymes.7. Effect of modulators and inhibitors.


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This equation predicts how

velocity of reaction(v) is related

to substrate concentration (S) if

enzyme concentration is held

constant.

v = Vmax(S)/Km+(S)

Km is the substrate concentration

at which velocity (v) is Vmax.

Plot of velocity versus (S) is

hyperbolicgraph Justlikeoxygen

myoglobindissociationcurve.

THE EFFECT OF SUBSTRATE CONCENTRATIONON REACTION VELOCITY

MICHAELIS-MENTEN EQUATION????

Q: for a fixed amount ofenzyme, what happens ifyou keep adding more andmore substrate?


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Is called as double

reciprocal plot because it is

a plot between reciprocals

of velocity of reaction(v)

and substrate concentration(S).

This plot is a straight line.

Slope of this graph is

Km/Vmax. The Y-intercept of the slope

is 1/Vmax.

The X-intercept of the slope

is -1/Km.

LINEWEAVER-BURKPLOT

(DOUBLE RECIPROCAL PLOT).


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INHITION OF ENZYME ACTIVITY

Competitiveinhibition

(competitive inhibitors

are substrate analogs

that compete with the

substrate for the active

sites of the enzyme ).

Non-competitive

inhibition(non-competitive inhibitors

bind at a site different

from the active site)


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QUESTION

What will be the effect of competitive and

non-competitive inhibition on the graphs

between v and (S); and graph between 1/v

and 1/(S)??????


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DIFFERENCE BETWEEN

Competetiveinhibition

Reversible.

Inhibitor and substrate

resemble. Inhibitor bind the active site.

Vmax is same.

Km is increased.

Inhibitor cannot bind with ES

complex. Lowers substrate affinity to

enzyme.

Complex is E-I.

Non-competetiveinhibition

Not reversible.

Iand S dont resemble.

binds other than active site.

Vmax decreases.

Km does not change.

Inhibitor Can bind with E-S

complex.

Substrate affinity remains

constant.

Complex is E-I or E-I-S.


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EXAMPLES OF COMPETETIVE

INHIBITION IN BIOLOGICAL SYSTMEM

Statins (HMG Co-A Reductase inhibitor).

Allopurinol (used to treat gout).

Sulphonamides antibiotics (e.g. septran). Beta-lactam antibiotics (e.g. penicillins).

ACE inhibitors (captopril,analpril and lisinopril).

Anti-cholinesterase drugs ( e.g. Physostigmineand neostigmine).

Warfarin.


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Sulfa Drug Is Competitive Inhibitor

-COOHH2N-

-SONH2H2N-

Precursor Folicacid

Sulfa drug (anti-inflammatory drug)

Para-aminobenzoic acid (PABA)

Bacteria needs PABA for

the biosynthesis of folic acid

Sulfa drugs has similar

structure with PABA, and

inhibit bacteria growth.


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Heavy metal inhibition of enzymes.

e.g. lead can inhibit ALA- synthase and

ferrochelatase enzyme in heme synthesisleading to anemia.

EXAMPLES OF NON-COMPETETIVE

INHIBITION IN BIOLOGICAL SYSTEM


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REGULATION OF ENZYME

ACTIVITY Allosteric regulation. Regulation of enzyme activity by covalent

regulation. Induction and repression of enzyme synthesis

by altering gene expression (by hormonal

action).


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A low molecular weightsubstance (theeffectoror

modifier) binds to the enzyme at

a specific site other than the

active site ( allostericsite) and

increases or decreases its activity

its activity (positiveandnegativeeffectors respectively).

Allosteric enzymes usually have

more than one subunit and more

than one active site.

In enzymes with multiple activesites that interact cooperatively,

velocty (v) versus (S) plot is

sigmoidal in shape just like

oxygen hemoglobin dissociation

curve.

Sigmoidal curve means binding of

one substrate molecule facilitates or

co-operates the binding of substrate

at other sites (co-operativebinding).

When substrate itself acts as an

effector the effect is called as

homotropic. If the effector is different from the

substrate the effect is called as

heterotropic.

Example 1: Heterotropic effetors

(phosphofructokinase is allosterically

inhibited by citrate). Example 2: Hexokinase enzyme is

allosterically inhibited by glucose-6

phosphate.

ALLOSTERIC REGULATION


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R

T

[S]

vo

Mechanism and Example of Allosteric Effect

SS

R

R

SS

A

[S]

vo

(+)

Allosteric site

Homotropic

(+)

Heterotropic

(+)

Allosteric site

Kinetics CooperationModels

(-)

(+)

(+)


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REGULATION BY CO-VALENT

MODIFICATION

This is usually done by phosphorylation and

dephosphorylation of the enzymes.

Example 1: Glycogen phosphorylase (theenzyme that breaks down glycogen) is

activated by phosphorylation.

Example2: Glycogen synthase (the enzyme

that synthesizes glycogen from glucose) is

activated by dephosphorylation.


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INDUCTION ANDREPRESSION OF

ENZYME ACTIVITY

This is a long term process and is relatively

slow to act.

Usually required for growth or at otherimportant physiologic events e.g. pregnancy.

This process is usually hormone dependent

which act on DNA to affect its transcription

into RNA.


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ENZYMES IN CLINICAL DIAGNOSIS

Alteration of plasma enzyme level in disease state. Plasma enzymes as diagnostic tools.

Isoenzymes and diseases of the heart(CK, TROP-T and TROP-I).

Amylase: raised in parotitis and acute pancreatitis.

Acid phosphatase: diagnostic for prostatitis and prostatecancer.

Alkaline phosphatase (ALP): diagnostic for obstructive

jaundice.

Gamma glutamyl tranferase:diagnostic for obstructivejaundice.

ASTand ALT:these are diagnostic for acute hepatitis.

LDH: Is diagnostic for acute myocardial infarction.

CPK: Is also diagnostic for acute MI.


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ENZYMES USEDTODIAGNOSE HEART

ATTACK

CPK-MB: peaks within 24 hours and usually comes

back to normal before 48 hours.

TROP-T and TROP-I(Newestmarkersfordiagnosisof

MI): These enzymes peak within 8-28 hours afterheart attack and remain elevated for 3-10 days.

AST or SGOT: peaks at 24-48 hours and may fall back

to normal by 72 hours.

LDH-1: Peaks at 3-4 days and remains elevated for

10-14 days.


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ENZYMES AS LABORATORY REAGENTS

Glucose oxidase: to detect glucose in urine

and blood).

Uricase: to detect uric acid in blood and urine Urease: to detect urea in blood and urine.


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THERAPEUTIC USES OF ENZYMES

Streptokinase and urokinase (for lysis of blood clot

e.g. after heart attack).

L-asparaginase (for the treatment of lymphoblastic

leukemia).

Digestiveenzymes as supplements in cases of

digestive disorders e.g. amylase, lipase,and protease.

Trypsin

and Chymotrypsin

(for degradation ofnecrotic tissue).

Hyaluronidase (for better absorption of ointments).


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Enzymes Biochemistry by Dr. Mudassar Ali Roomi

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Enzymes Biochemistry by Dr. Mudassar Ali Roomi