1.
PRINCIPLES OF
ENZYME ACTIVITY

2.
MECHANISMS OF ENZYME ACTION
• ENZYMES ENHANCE THE RATE OR VELOCITY OF A REACTION BUT
DO NOT ALTER THE EQUILIBRIUM CONSTANT
• CATALYSIS IS BROUGHT ABOUT IN ONE OR MANY WAYS
LOWERING OF ACTIVATION ENERGY
• ACTIVATION ENERGY IS THE ENERGY REQUIRED TO RAISE THE
LEVEL OF THE SUBSTRATE FROM THE GROUND TO THE
TRANSITION STATE
• ENZYMES REDUCE THE MAGNITUDE OF THIS ACTIVATION
ENERGY
Acid hydrolysis – 26,000 cal/mol
SUCROSE
Sucrase- 9,000 cal/mol

3.
without enzyme
with enzyme
E
time
• THIS CAUSES REACTIONS TO PROCEED AT A LOWER
TEMPERATURE ADVANTAGEOUS IN BIOLOGICAL SYSTEMS

4.
CATALYSIS BY PROXIMITY (ENTROPY EFFECT)
• ENZYMES DECREASE THE ENTROPY OF THE REACTANTS
• ENAABLES THE REACTANTS TO COME CLOSER TO THE ENZYME
• ENHANCES THE RATE OF ES FORMATION
CATALYSIS BY STRAIN
• FOLLOWS THE INDUCED FIT THEORY
• SUBSTRATE IS STRAINED DUE TO THE CONFORMATIONAL CHANGE
IN THE ENZYME
• ENERGY LEVEL OF SUBSTRATE IS RAISED DUE TO THE STRAIN
• COVALENT BONDS ARE BROKEN
• Eg. LYSOZYME CLEAVES β-1,4 GLYCOSIDIC BONDS

5.
ACID-BASE CATALYSIS
• AMINO ACID RESIDUES AT ACTIVE SITE OF ENZYME ACT AS
ACIDS/BASES i.e. DONOR OR ACCEPTOR OF H⁺/e⁻
• Acids NH₂‚ –OH‚ –SH‚ ϵ - amino groups
• Bases -COO⁻ , conjugates of acidic groups
• Eg. CLEAVAGE OF PHOSPHODIESTER BONDS BY RIBONUCLEASE
COVALENT CATALYSIS
• ENZYMES HAVE -vely CHARGED (NUCLEOPHILIC) OR +vely
CHARGED (ELECTROPHILIC) GROUPS AT ACTIVE SITE
• THESE GROUPS FORM TRANSIENT COVALENT BONDS WITH THE
SUBSTRATE
• ENZYME ITSELF BECOMES A REACTANT FOR A TRANSIENT PERIOD
OF TIME
• COVALENT BONDS ARE BROKEN TO FORM PRODUCTS
• Eg. CLEAVAGE OF PEPTIDE BONDS BY TRYPSIN

6.
FACTORS AFFECTING ENZYME ACTIVITY
CONCENTRATION OF SUBSTRATE
• INCREASE IN SUBSTRATE CONCENTRATION GRADUALLY
INCREASES THE RATE/VELOCITY OF AN ENZYMATIC REACTION
Vmax C
B
V ½ Vmax
A
km
k₁ k₃ [S]
E+S ES E+P
k₂
k₂+ k₃
k₁ =Km

7.
Vmax[S]
V= (Michaelis-Menten equation)
Km+ [S]
If V= ½ Vmax
Vmax [S]
Then, ½ Vmax =
Km+ [S]
Km= [S]
i.e. at ½ Vmax , Km=[S]
Km IS DEFINED AS THE SUBSTRATE CONCENTRATION AT HALF
MAXIMAL VELOCITY (moles/L)

8.
SIGNIFICANCE OF Km VALUE
• IT IS CONSTANT & IS THE CHARACTERISTIC FEATURE (SIGNATURE)
FOR A GIVEN ENZYME
• IT IS INDEPENDENT OF ENZYME CONCENTRATION
• IT INDICATES THAT HALF OF ENZYME MOLECULES ARE BOUND
WITH THE SUBSTRATE MOLECULES AT THAT PARTICULAR
SUBSTARTE CONCENTRATION
• Km DENOTES THE AFFINITY OF ANY ENZYME FOR ITS SUBSTRATE
• LOW Km VALUE INDICATES A STRONG AFFINITY BETWEEN AN
ENZYME & ITS SUBSTRATE WHEREAS A HIGH Km VALUE REFLECTS
WEAK AFFINITY.

9.
CONCENTRATION OF ENZYME
V
[E]
• AS THE CONCENTRATION OF ENZYME IS INCREASED THE
VELOCITY/RATE OF REACTION INCREASES PROPORTIONATELY
(when all other factors affecting enzyme activity are kept
constant)
• THIS PROPERTY IS USED FOR DETERMINING THE ACTIVITY OF
ENZYMES FOR DIAGNOSIS OF DISEASES

10.
TEMPERATURE
V optimum temperature
0 10 20 30 40 50 60
Temp
• INCREASE IN TEMP. RESULTS IN AN INCREASE IN THE RATE OF RXN UPTO A
CERTAIN EXTENT AFTER WHICH THE ACTIVITY DECREASES
• MAXIMUM ACTIVITY IS SEEN AT THE OPTIMUM TEMPERATURE

11.
pH
1 2 3 4 5 6 7 8 9 10 11 12 13 14
pH
• MAXIMUM ACTIVITY OF ENZYME IS SEEN AT THE OPTIMUM pH ,
BELOW AND ABOVE WHICH THE ACTIVITY DECLINES
• H⁺ IONS ALTER THE IONIC CHARGES ON THE AMINO ACIDS AT THE
ACTIVE SITE
• ALSO AFFECT THE IONISATION OF SUBSTRATE AND ES COMPLEX
• DISSOCIATION OF CO-ENZYMES AT EXTREME pH
• DISRUPTION OF IONIC & HYDROGEN BONDS

12.
CONCENTRATION OF PRODUCTS
• IN CONCENTRATION OF PRODUCT es THE RATE OF RXN
• PRODUCT FORMS A LOOSE COMPLEX WITH THE ENZYME AT
THE ACTIVE SITE & PREVENTS THE BINDING OF SUBSTRATE,
THUS DECREASINGS THE ACTIVITY OF ENZYME
• END PRODUCT INHIBITION IN BIOLOGICAL SYSTEMS IS
IMPORTANT FOR REGULATION OF METABOLIC PATHWAYS
E₁ E₂ E₃
A B C D
-

13.
ACTIVATORS & INHIBITORS
• PRESENCE OF IONS (Mg²⁺, Mn²⁺, Zn²⁺,Ce⁻) es THE ACTIVITY OF
CERTAIN ENZYMES
• PRESENCE OF INHIBITIORS es THE ACTIVITY
• IONS COMBINE WITH SUBSTRATE, FORM METAL-ES COMPLEX,
PARTICIPATE DIRECTLY IN RXNS, CHANGE CONFORMATION OF
ENZYME, INHIBIT SUBSTRATE BINDING

14.
ENZYME INHIBITION

15.
ENZYME INHIBITION
ENZYME INHIBITORS ARE MOLECULAR AGENTS THAT INTERFERE
WITH CATALYSIS (SLOW DOWN OR HALT REACTIONS) AND
REDUCE THE RATE OF REACTION
Eg. ASPIRIN – Pharmaceutical agent
TYPES OF INHIBITION
REVERSIBLE
COMPETITIVE
NON-COMPETITIVE
MIXED
UN-COMPETITIVE
IRREVERSIBLE

16.
REVERSIBLE INHIBITION
ACTIVITY OF ENZYME IS RESTORED FULLY WHEN THE INHIBITOR
IS REMOVED FROM THE SYSTEM
E + I EI
Ki inhibition constant
Competitive inhibition
 Inhibitor competes with substrate
 ″ is a structural analog of substrate
 ″ binds to the enzyme at SBS
 Both ES & EI complex formed
 IF [S] >> [I] normal Vmax
 IF [S] << [I] [ES] Vmax
 MORE OF SUBSTRATE IS NEEDED TO FORM ES APPARENT in
Km

17.
Non-Competitive inhibition
 NO COMPETITION BETWEEN ʽSʼ & ʽIʼ
 STRUCTURALLY DIFFERENT
 ʽIʼ BINDS TO A SITE OTHER THAN SBS
 EI AND ESI COMPLEX IS FORMED
 AFFINITY OF ʽSʼ FOR ʽEʼ UNALTERED
 Km REMAINS SAME
 ESI COMPLEX BREAKDOWN IS SLOWED
 REACTION IS SLOWED Vmax

18.
Un-competitive inhibition
 RARE , ONLY SEEN IN BISUBSTRATE RxNS.
 ʽΙʼ BINDS TO ES COMPLEX
 ʽIʼ HAS NO AFFINITY FOR FREE ENZYME
 ʽESIʼ COMPLEX FORMED
 Km apparent (as [S] required to reach ½ Vmax decreases by
factor ά )

19.
Mixed inhibition
 ʽESʼ, ‘EI’ & ʽΕSΙʼ FORMED
 BOTH Km & Vmax ALTERED ( Km; Vmax)
 USUALLY SEEN IN BI-SUBSTRATE RxNS.

20.
INHIBITION Vmax Km
Competitive same
Non-competitive same
Un-competitive
Mixed

21.
IRREVERSIBLE INHIBITION
 ‘I’ BINDS COVALENTLY TO ʽEʼ
 NO COMPETITION BETWEEN ʽIʼ & ʽSʼ
 DESTRUCTION OF FUNCTIONAL GROUP AT ACTIVE SITE ESSENTIAL
FOR ACTIVITY
 NOT REVERSED BY REMOVING ʽIʼ OR INCREASING [S]
 KINETICS IS SIMILAR TO REV. NON-COMPETITIVE INHIBITION
 SUICIDE INHIBITION
 SPECIAL TYPE OF IRREVERSIBLE INHIBITION
 ʽIʼ BINDS TO SBS UNDERGOES CATALYSIS PRODUCT
FORMED IS IRREVERSIBLE INHIBITOR BINDS COVALENTLY
& DESTROYS ʽEʼ

22.
APPLICATIONS OF ENZYME INHIBITION
Inhibitor Enzyme Use
Allopurinol Xanthine oxidase Gout
Methotrexate DHF reductase Cancer
Succinylcholine Ach esterase Muscle relaxant
Lovastatin HMG CoA reductase Atherosclerosis