Dr. Riddhi Datta

ENZYMES

Dr. Riddhi Datta

Enzymes

• A biomolecule, either protein or RNA, that catalyzes a specific biochemical reaction is called enzyme.
• Holoenazyme: A complete, catalytically active enzyme together with its bound cofactor is called holoenzyme.
• Cofactor: Some enzymes require additional inorganic components like metal ions or complex organic or metalorganic molecule for its catalytic activity. This non-protein part is called cofactor.
• Apoenzyme: The protein part of the enzyme with no co-factor bound is called apoenzyme.

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Dr. Riddhi Datta

Enzymes

Dr. Riddhi Datta

Enzymes

• Prosthetic group: A cofactor that is tightly or covalently bound to the apoenzyme is called prosthetic group. They cant be separated by dialysis.

Ex: Succinate dehydrogenase and FAD

• Coenzyme: A cofactor that is loosely bound to the apoenzyme is called coenzyme. They are small heat stable organic molecules that can be separated by dialysis.

Ex: Lactic dehydrogenase and NADH₂

• Metal activator: Metallic mono- or divalent cations that bind to and activate the apoenzyme are called metal activators.

Ex: Carbonic anhydrase and Zn²⁺

• Metalloenzymes: Enzymes where metal ions are permanently bound to the apoprotein and remain associated even after reaction is over.

Ex: Cytochrome oxidase with Fe²⁺

Dr. Riddhi Datta

How Enzymes Work

• An enzyme provides a specific environment within which a given reaction can occur more rapidly.
• An enzyme-catalyzed reaction takes place within the confines of a pocket on the enzyme called the active site.
• The molecule that is bound in the active site and acted upon by the enzyme is called the substrate.
• The surface of the active site is lined with amino acid residues with substituent groups that bind the substrate and catalyze its chemical transformation. These residues are called the catalytic groups.

Dr. Riddhi Datta

E:enzyme

S:substrate

P: product

ES: transient enzyme-substrate complex

EP: transient enzyme-product complex

Enzymes increase the rate of a reaction and do not affect reaction equilibria.

Dr. Riddhi Datta

Active sites of enzymes have some common features

• The active site is formed by groups that come from different parts of the amino acid sequence.

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• The active site takes up a relatively small part of the total volume of an enzyme.

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• Active sites are three-dimensional clefts or crevices.

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• Substrates are bound to enzymes by multiple weak attractions like electrostatic interactions, hydrogen bonds, van der Waals forces, and hydrophobic interactions.

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• The specificity of binding depends on the precisely defined arrangement of atoms in an active site.

Dr. Riddhi Datta

• The enzyme and the substrate possess specific complementary geometric shapes that fit exactly into one another.

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• The model was proposed by Emil Fischer in 1890.

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• It does not explain the stabilization of the transition state that the enzymes achieve.

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• It is an obsolete model.

Lock-and-key model of enzyme-substrate binding

Dr. Riddhi Datta

Induced-Fit Model of Enzyme-Substrate Binding

• The enzyme undergoes a change in conformation when the substrate binds, induced by multiple weak interactions with the substrate.

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• The active site forms a shape complementary to the substrate only after the substrate has been bound.

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• The induced fit model shows that enzymes are rather flexible structures. 

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• It was proposed by Daniel Koshland in 1958.

Dr. Riddhi Datta

• The starting point for either the forward or the reverse reaction is called the ground state.

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• The free-energy change for this reacting system at pH 7.0 is expressed as biochemical standard free energy change (∆G’°).

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• A reaction can occur spontaneously only if ∆G’° is negative: exergonic reactions

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• A system is at equilibrium and no net change can take place if ∆G’° is zero.

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• A reaction cannot occur spontaneously if ∆G’° is positive and input of free energy is required: endergonic reactions

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• There is an energy barrier between S and P.

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• To undergo reaction, the molecules must overcome this barrier and must be raised to a higher energy level.

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• This is called the transition state.

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• The difference between the energy levels of the ground state and the transition state is the activation energy, ∆G^‡.

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• The rate of a reaction reflects this activation energy: a higher activation energy corresponds to a slower reaction.

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Dr. Riddhi Datta

• Catalysts (enzymes) enhance reaction rates by lowering activation energies.

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• Enzymes accelerate the inter-conversion of S and P.

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• The enzyme is not used up in the process, and the equilibrium point is unaffected.

Dr. Riddhi Datta

Enzymes inhibitors

• Enzyme inhibitors are molecular agents that interfere with catalysis, slowing or halting enzymatic reactions.

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• Example: aspirin (acetylsalicylate) inhibits the enzyme that catalyzes prostaglandins synthesis which produce pain.

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• Enzyme inhibition can be either reversible or irreversible.

Dr. Riddhi Datta

• An irreversible inhibitor dissociates very slowly from its target enzyme

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• It binds tightly to the enzyme, either covalently or non-covalently.

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• Some irreversible inhibitors are important drugs.

• Reversible inhibition is characterized by a rapid dissociation of the enzyme inhibitor complex.

Irreversible inhibitors

Reversible inhibitors

Dr. Riddhi Datta

Competitive Inhibition

• A competitive inhibitor competes with the substrate for the active site of an enzyme.

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• The inhibitor (I) resembles the geometry of the substrate.

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• It occupies the active site forming EI complex and prevents substrate binding to the enzyme.

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• A competitive inhibitor diminishes the rate of catalysis by reducing the proportion of enzyme molecules bound to a substrate.

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• At any given inhibitor concentration, competitive inhibition can be relieved by increasing the substrate concentration.

Dr. Riddhi Datta

Uncompetitive Inhibition

• An uncompetitive inhibitor binds at a site distinct from the substrate active site.

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• It binds to the ES complex forming the ESI complex.

• An uncompetitive inhibitor acts by decreasing the turnover number.

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• Uncompetitive inhibition cannot be overcome by increasing the substrate concentration.

Dr. Riddhi Datta

• A mixed inhibitor binds at a site distinct from the substrate active site.

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• It binds to either E or ES.

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• A single inhibitor both hinders the binding of substrate and decreases the turnover number of the enzyme.

Mixed Inhibition

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Effects of reversible inhibitors on apparent V_max and apparent K_m

Dr. Riddhi Datta

Feedback inhibition

• In some multienzyme systems, the regulatory enzyme is specifically inhibited by the end product of the pathway whenever the concentration of the end product exceeds the cell’s requirements.

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• This type of regulation is called feedback inhibition.

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• Buildup of the end product ultimately slows the entire pathway.

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• Allosteric enzymes function through reversible, noncovalent binding of regulatory compounds called allosteric modulators or allosteric effectors to a site other than active site.

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• Allosteric modulators are generally small metabolites or cofactors.

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• The conformational changes induced by the modulator(s) interconvert more-active and less-active forms of the protein.

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• The modulators for allosteric enzymes may be inhibitory or stimulatory.

Allosteric enzymes

Dr. Riddhi Datta

• The substrate binding site is on the catalytic subunit (C) and the modulator binding site is on the regulatory (R) subunits

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• Binding of the positive (stimulatory) modulator (M) to its specific site on the regulatory subunit causes a conformational change,

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• This change activates the catalytic subunit.

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• On dissociation of the modulator from the regulatory subunit, the enzyme reverts to its inactive or less active form.