enzymes: catalytic strategies & regulatory mechanisms harini chandra binding of a substrate to...

Enzymes: Catalytic strategies & regulatory mechanisms

Harini Chandra

Binding of a substrate to an enzyme catalytic site brings about conformational changes in the enzyme with multiple weak interactions being formed with the

substrate molecule. The amino acid side chains of the enzyme aid in cleavage and formation of bonds thereby providing a variety of mechanisms for catalysis to occur.

Master Layout (Part 1)

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1 This animation consists of 4 parts:Part 1 – Covalent catalysisPart 2 – Acid-base catalysisPart 3 – Metal ion catalysisPart 4 – Regulatory strategies

A B

X

A X B

A X B

H2O

Substrate

Nucleophilic group

Covalent bond

Product Product

Source: Modified from Biochemistry by A.L.Lehninger et al., 4 th edition (ebook)

Definitions of the components:Part 1 – Covalent catalysis

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11. Covalent catalysis: A catalytic strategy wherein the active site of an enzyme is lined with a reactive group, usually a powerful nucleophile, that undergoes temporary covalent modification during the course of the reaction. A transient covalent bond between the enzyme and substrate facilitates catalysis by providing a pathway with lower activation energy.

2. Substrate: The molecule(s) present at the beginning of a reaction that are modified by enzyme are known as substrate(s). An enzymatic reaction may have one or more substrates depending upon the reaction.

3. Covalent bond: A chemical bond formed between atoms of two reacting species that involves the sharing of outer orbital electrons for bond formation.

4. Nucleophilic group: An electron rich group that readily attacks a positively charged centre to form a chemical bond by donating its electrons. The term nucleophile translates to “nucleus lover”.

5. Product: The molecules produced as a result of an enzymatic reaction are known as the products. A reaction may yield one or more products with the enzyme being regenerated at the end of the reaction.

Part 1, Step 1:

Action Audio Narration

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4 Description of the actionAs shown in animation.

First show ‘A’ and ‘B’ joined by thick line as shown along with ‘X’. ‘X’ must be shown to attack ‘A’ and when this happens, the line between A&B must become a dotted line and a new dotted line must appear between A&X. Next, B must move away and the dotted line must disappear while the dotted line between A&X must become a thick line. Next the blue ‘H2O’ must appear and must pass through the line between A&X. When this happens, the line between A&X must break and they must move apart from each other.

Covalent catalysis involves the formation of a transient covalent bond between the nucleophile present in the enzyme and the substrate molecule. Formation of this bond provides an alternative reaction pathway that has lower activation energy than the uncatalyzed reaction. Several amino acid side chains act as effective nucleophiles that facilitate the reaction. The enzyme is regenerated in its unaltered form at the end of the reaction.

A B

H2O

Substrate

X

Nucleophilic group

Covalent bond

Products

Regenerated catalyst

Covalent catalysis


Part 1, Step 2:


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4Description of the action

As shown in animation.

First show the ‘enzyme’ and ‘substrate’. Then show the black arrow appearing after which the grey arrow must appear followed by removal of the group ‘XH’ as shown and appearance of the figure after the arrow. The red line shown must then appear. The blue oval marked ‘H2O’ must then appear followed by the black arrow as shown. This must be followed by appearance of the downward grey arrow and the figures shown below it.

Chymotrypsin is one such enzyme that carries out catalysis by covalent modification. It possess a catalytic triad of histidine, aspartic acid and serine at its active site with the serine at position 195 serving as a highly powerful nucleophile. The reaction between the serine hydroxyl group and the unreactive carbonyl group of the substrate helps in bringing about product formation with regeneration of the enzyme after the reaction. Covalent modification of this serine residue led to irreversible inactivation of the enzyme, which clearly suggested that it performs a vital role in catalysis.

Example for covalent catalysis - Chymotrypsin

X C

O

R

Substrate

C

O

RO

Ser-195XH

H2O

Ser-195

OH C

O

R

HO

OH

Ser-195

Enzyme

Regenerated enzyme

Product

Source: Modified from Biochemistry by Lubert Stryer et al., 6 th edition (ebook)


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R OH C O

R1

N R2

H

C OR OH

R1

N R2

H

B

C OR O

R1

N R2

HBH

C OR O

R1

N R2

H

H

N R2

H

H

C OR O

R1Substrates

Base

Intermediate

Products

Definitions of the components:Part 2 – Acid-base catalysis

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11. Acid-base catalysis: Biochemical reactions involving the formation of unstable charged intermediates are often stabilized by transfer of protons to or from the substrate or intermediate. In case of enzyme catalyzed reactions, weak proton donors or acceptors are often present as amino acid side chains at the active site of the enzyme itself. These groups mediate proton transfer reactions which provide rate enhancement of several orders of magnitude.

2. Substrate(s): The molecules present at the beginning of a reaction that are modified by means of the enzyme are known as substrates. An enzymatic reaction may have one or more substrates depending upon the reaction.

3. Enzyme: The biocatalyst responsible for bringing about an increase in the rate of reaction for conversion of substrate to product.

4. Intermediate: Enzymatic reactions proceed through formation of a transition state i.e. an intermediate state in between substrate and product having higher free energy than that of either the substrate or product. The transition state is the least stable species of the reaction pathway due to its high free energy and is therefore the most seldom occupied.

5. Base: An aqueous solution or substance that can accept hydronium ions by donating its free electron pair is known as a base.

6. Product(s): The molecules produced as a result of an enzymatic reaction are known as the products. A reaction may yield one or more products with the enzyme being regenerated at the end of the reaction.

Part 2, Step 1:


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First show the two ‘substrates’ on top. The green circle containing ‘OH’ group must move towards the yellow circle containing ‘C’ as shown. The down arrow must then appear to give the figure below.

Biochemical reactions involving the formation of unstable charged intermediates are often stabilized by transfer of protons to or from the substrate or intermediate. For non-enzymatic reactions, acid-base catalysis may involve only the hydronium or hydroxyl ions of water, referred to as specific acid-base catalysis.

R OH

C O

R1

N R2

HSubstrates

C OR OH

R1

N R2

H Intermediate

Acid-base catalysis


Part 2, Step 2:


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Next, show the blue circle ‘base’ appearing which must move towards the green circle containing ’OH’ as depicted. It must then remove the red ‘H’ from it and the figure below must appear. The blue circle must then move away.

In many cases, however, water alone does not suffice to catalyze the reaction. In such cases, proton transfer is facilitated by weak organic acids or bases. Organic acids act as proton donors while organic bases can serve as proton acceptors.

C OR OH

R1

N R2

H

BBase

C OR O

R1

N R2

H

BH

Acid-base catalysis


Part 2, Step 3:


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Next, the blue circle must move towards the pink circle containing ‘N’. The red ‘H’ must then be transferred to this group as shown in the figure below. The blue circle must then move away.

In case of enzyme catalyzed reactions, weak proton donors or acceptors are often present as amino acid side chains at the active site of the enzyme itself. The precise positioning of these groups within the active site mediate proton transfer reactions which can provide rate enhancements of several orders of magnitude.

C OR O

R1

N R2

H

BH

C OR O

R1

N R2

H

H

B

Acid-base catalysis


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Show the arrows appearing in the figure above at the positions indicated followed by the downward arrow and the ‘products’ below.

Acid-base catalysis is a common mechanism of action employed by many enzymes. It is often used in combination with another mechanism such as covalent catalysis. The ease of stabilization of charged intermediates by the amino acid side chains helps in lowering the activation energy for product formation.

C OR O

R1

N R2

H

H

N R2

H

H

C OR O

R1

Products

Acid-base catalysis


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(PLEASE RE-DRAW ALL FIGURES.)First show the top left figure appearing with its labels followed by appearance of the arrow and the compound above it. Next the second figure on the right must appear followed by the downward arrow and the figure below it.

Chymotrypsin is one such enzyme that employs both covalent catalysis as well as acid-base mechanism. The arrangement of the catalytic triad, consisting of Aspartic acid, Histidine and Serine, at the enzyme’s active site is such that the histidine residue serves as a general base catalyst by polarizing the hydroxyl group of serine. The alkoxide ion thus generated in the serine residue makes it a more powerful nucleophile.

Example for acid-base catalysis - Chymotrypsin

Chymotrypsin enzyme

Active site – catalytic triad

Asp 102

His 57

Ser 195

Substrate

Oxyanion hole

Tetrahedral intermediate

Source: Biochemistry by Lubert Stryer et al., 6 th edition (ebook)

Part 2, Step 6:


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(PLEASE RE-DRAW ALL FIGURES.)In continuation with the previous slide. The grey arrow must then appear after ‘tetrahedral intermediate’ followed by the figure on the right. Next the arrow must appear along with the curved arrow showing removal of ‘product 1’ followed by the figure below.

Following substrate binding and nucleophilic attack of the serine on the carbonyl group, the geometry of the intermediate becomes tetrahedral and the negative charge developed on the carbonyl oxygen gets stabilized through interactions with other side chains of the proteins, in a site known as the oxyanion hole. An internal proton transfer then causes the tetrahedral intermediate to collapse and generate the acyl-enzyme intermediate after which the amine group is released from the active site.


Acyl-enzyme intermediate


Product 1



Part 2, Step 7:


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(PLEASE RE-DRAW ALL FIGURES.)In continuation with the previous slide. The reaction arrow must appear along with entrance of a molecule of ‘H2O’ as shown. This is followed by appearance of the figure on the right after which the downward arrow must appear along with the figure below.

Once the amine group leaves the enzyme’s active site, it is replaced by a molecule of water which carries out hydrolysis of the ester group of the acyl-enzyme intermediate. Mechanism for hydrolysis again proceeds via formation of a tetrahedral intermediate with histidine acting as a general acid catalyst and the negative charge on oxygen being stabilized by residues in the oxyanion hole.

Example for acid-base catalysis - ChymotrypsinAcyl-enzyme intermediate

Oxyanion hole




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(PLEASE RE-DRAW ALL FIGURES.)In continuation with the previous slide. The arrow must then appear to show the figure on the right top followed by the downward arrow and removal of the compound shown as ‘product 2’ followed by the appearance of the figure below.

The tetrahedral intermediate then breaks down to liberate the second product in the form of a carboxylic acid along with regeneration of the enzyme which is then ready for another round of catalysis. Internal proton transfers between amino acid side chains therefore play a vital role in acid-base catalysis by enzymes.


Tetrahedral intermediateProduct 2

Regenerated enzyme



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1


This animation consists of 4 parts:Part 1 – Covalent catalysisPart 2 – Acid-base catalysisPart 3 – Metal ion catalysisPart 4 – Regulatory strategies

M2+ O

H

H

H

M2+ O

H

C O

R1

R2

M2+ O

H

C O

R1

R2

H2O

O

H

C O

R1

R2

Product

Metal ion

IntermediateRegenerated catalyst

Substrate

M2+

H2 O

Definitions of the components:Part 3 – Metal ion catalysis

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11. Metal-ion catalysis: Metal ions, either present in solution or bound to the enzyme itself, facilitate catalysis by forming favorable interactions that orient the substrate and enzyme in suitable positions for transition state, and subsequently, product formation. These interactions help in stabilizing the intermediates formed, thereby lowering the activation energy required for reaction to occur.

2. Substrate(s): The molecules present at the beginning of a reaction that are modified by means of the enzyme are known as substrates. An enzymatic reaction may have one or more substrates depending upon the reaction.

3. Enzyme: The biocatalyst responsible for bringing about an increase in the rate of reaction for conversion of substrate to product.

4. Intermediate: Enzymatic reactions proceed through formation of a transition state i.e. an intermediate state in between substrate and product having higher free energy than that of either the substrate or product. The transition state is the least stable species of the reaction pathway due to its high free energy and is therefore the most seldom occupied.

5. Metal ion: Metals usually present in their divalent state (i.e. +2 charge) facilitate catalysis. One or more metal ion has been found to be required for catalysis of nearly one third of all enzymatic reactions. One of the most commonly found metal ions in enzymatic mechanisms is Zn2+.

6. Product(s): The molecules produced as a result of an enzymatic reaction are known as the products. A reaction may yield one or more products with the enzyme being regenerated at the end of the reaction.

Part 3, Step 1:


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First show the purple oval along with the ‘water’ figure. The purple circle must then approach the blue circle of water. When this happens, the red line must appear between the two and one green ‘H’ must leave in the form of ‘H+’ as depicted to give the figure on the right.

Metal ions, either present in solution or bound to the enzyme itself, facilitate catalysis by forming favorable interactions between enzyme and substrate or in the transition state. Metal ions in the active site of the enzyme typically react with a water molecule and activate it by facilitating generation of a strong nucleophile in the form of a hydroxide ion.

H

O

H

H

M2+ O

H

M2+

Metal ion

WaterStrong nucleophile

generated

Metal ion catalysis


Part 3, Step 2:


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In continuation with previous slide. Show the ‘substrate’ molecule appearing after which the black arrows must appear as shown. The grey arrow must then appear along with the figure on the right.

The nucleophilic alkoxide ion attacks the unreactive carbonyl group to form a tetrahedral intermediate in which the charges are stabilized by the metal ion. These favorable interactions help in orienting the substrate and enzyme in suitable positions for transition state, and subsequently, product formation.

C O

R1

R2

Substrate

M2+ O

H

Strong nucleophile generated

Metal ion catalysis

M2+ O

H

C O

R1

R2

Intermediate


Part 3, Step 3:


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In continuation with previous slide. Show the blue ‘H2O’ appearing which must move towards the violet oval as shown. This is followed by appearance of the small black arrow after which the grey arrow and the figures on the right must appear as depicted.

Hydrolysis of the stabilized intermediate leads to product formation along with liberation of the regenerated metal catalyst. Thus by lowering the activation energy of the transition state, metal ions facilitate enzyme catalyzed reactions. Almost one third of known enzymes have been found to require one or more metal ions for their activity.

Metal ion catalysis

M2+ O

H

C O

R1

R2

Intermediate

O

H

C O

R1

R2

H2O

M2+

H2 O

ProductRegenerated catalyst


Part 3, Step 4:


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First show appearance of ‘carbonic anhydrase’, ‘active site’ and ‘water’. This is followed by appearance of the black arrow mark as shown after which the grey arrows, the figure on the right and the green ‘H+’ must appear.

Carbonic anhydrase is an enzyme responsible for hydration and dehydration reactions of carbon dioxide and bicarbonate respectively and has been found to have a divalent zinc ion associated with its activity. The zinc ion in its active site is bound to the imidazole rings of three histidine residues as well as to a molecule of water. This binding to water facilitates formation of the hydroxide nucelophile with concomitant release of a proton.

Example for metal ion catalysis – Carbonic anhydrase

Zn2+

His 119

His 96

His 94

O

H

HH

Zn2+

His 119

His 96

His 94

O

H

Carbonic anhydrase

Active site

Strong nucleophile generated

Water


Part 3, Step 5: 1

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Zn2+

His 119

His 96

His 94

O

H

C

O

OSubstrate

Zn2+

His 119

His 96

His 94

O

H

C

O

O

Electrostatic stabilization forces

Intermediate

Action Audio NarrationDescription of the actionAs shown in animation.

In continuation with previous slide. The ‘substrate’ should then appear after which the black arrows must be shown as depicted. This is followed by the grey downward arrow and the figure show below. The dotted red line must appear in the end after the figure is shown as depicted in animation.

The generated hydroxide ion at the active site then attacks the carbon dioxide substrate, converting it into a bicarbonate ion. The negative charge generated on the oxygen atom is stabilized by interactions with the zinc ion.


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Zn2+

His 119

His 96

His 94

O

H

C

O

O

O

H

H

Zn2+

His 119

His 96

His 94

O

H

H

C

O

O

O

HProduct

(bicarbonate)

Regenerated enzyme

Action Audio NarrationDescription of the actionAs shown in animation.

In continuation with previous slide. Another ‘water’ molecule shown above must then appear and approach the brown oval. The grey arrow must then appear followed by the figures shown on the right.

Binding of another molecule of water to the zinc ion at the active site of the enzyme leads to release of the bicarbonate ion and regeneration of the enzyme molecule ready for another round of catalysis. Several pH related studies have provided substantial proof for this mechanism.



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Enzyme regulatory strategies

Allosteric/ feedback inhibition

Isozyme forms

Reversible covalent

modification

Proteolytic activation


Definitions of the components:Part 4 – Regulatory strategies

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11. Allosteric/feedback inhibition: Enzymes that contain distinct catalytic and regulatory sites are said to be allosteric in nature. Binding of regulatory molecules at the regulatory sites triggers off a series of conformational changes that ultimately reach the active site and prevents binding of substrate molecule thereby regulating catalysis. In many pathways, the reaction that is regulated involves the enzyme catalyzing the first step of the pathway so that all further reactions taking place after the first also automatically get regulated due to lack of substrate availability. When this enzyme is regulated by the final product of the reaction pathway, it is known as feedback inhibition. Most allosteric enzymes are subject to feedback control, a popular example being aspartate transcarbamoylase.

2. Isozyme forms: Isoenzymes are homologous enzymes within a single organism that differ slightly in their structure but catalyze the same reaction. They are mostly expressed in different tissues and have differing kinetic parameters such as substrate affinity (Km) and maximum velocity (Vmax). These differing properties allow the isozyme forms to be differentially regulated at varying points of time. Example: Lactate dehydrogenase exists in different forms in heart and muscle tissue. The heart isozyme has a higher affinity for substrates and is allosterically inhibited by high levels of pyruvate while the muscle enzyme is not.

3. Reversible covalent modification: Several covalent modifications such as phosphorylation, uridylylation, methylation etc. significantly alter the catalytic activity of enzymes. Covalently modified enzymes may either have increased or decreased activity for the reaction it catalyzes. The enzymes responsible for covalent modification of other enzymes are usually regulated by further control mechanisms. Example: Enzymes of glycogen metabolism such as glycogen phosphorylase are regulated by covalent modification.

Definitions of the components:Part 4 – Regulatory strategies

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14. Proteolytic activation: Many enzymes exist in their inactive forms, known as zymogens, and need to be activated by hydrolysis of one of more peptide bonds. This removal of certain regulatory residues irreversibly converts the enzyme into its active form. The enzyme is then degraded after completion of catalysis. Example: Digestive enzymes such as trypsin, chymotrypsin as well as blood clotting factors are regulated by mechanism of proteolytic activation.

Part 4, Step 1:


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Activity of all enzymes must be regulated to ensure that they function only to the desired extent at the appropriate locations within an organism. Common mechanisms of regulation include allosteric or feedback inhibition, control of isozyme forms, reversible covalent modification and proteolytic activation. <If user clicks on any of these four tabs, the description as given in previous two slides must be narrated.>

(Please redraw figures.)The top heading must be shown followed by the four arrows and the headings in the circles below. The user should be allowed to click on any of the headings in the circles to view the description provided in the previous two slides. Another ‘proceed’ tab must be present on the right bottom and when user clicks on that, the text in the blue circle and yellow circle must be highlighted after which it must move to the next slide.

Enzyme regulatory strategies

Allosteric/ feedback inhibition

Isozyme forms

Reversible covalent

modification



Part 4, Step 2:


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Allosteric enzymes, which possess distinct regulatory and catalytic sites, are often found as the first enzyme of a reaction pathway. Regulation of the first enzyme of a pathway by the final product of the pathway is known as feedback inhibition. Binding of the regulator molecule to the regulatory site of the enzyme triggers a series of conformational changes that are ultimately transmitted to the active site where substrate binding is then inhibited. It has been observed that allosteric enzymes do not obey regular Michaelis-Menten kinetics.

First show blue ‘enzyme A’ along with green ‘substrate A’. Green substrate must bind in the pocket as shown after which its shape must change to a brown oval. This oval must leave the pocket after which the sequence of arrows and the other shapes must appear as depicted in animation. When ‘Substrate E’ appears, it must move towards ‘enzyme A’ and bind in the other pocket as depicted. When this happens, the pocket on top must change shape as shown and the green substrate should not be able to bind to it. Then red crosses must appear across all the other reaction arrows as well.

Allosteric/feedback inhibition

Enzyme A

Substrate ASubstrate B

Enzyme B

Substrate C

Enzyme C

Substrate D

Enzyme D

Substrate E

Allosteric enzymes DO NOT obey Michaelis-Menten kinetics.


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Aspartate transcarbamoylase, which catalyzes the first step of pyrimidine biosynthesis, is an allosteric enzyme having distinct regulatory and catalytic subunits. Binding of substrate to the catalytic subunits induces conformational changes that stabilize the relaxed state or R state of the enzyme, thereby facilitating the enzymatic reaction. The inhibitor for this enzyme is CTP which is the final product of the pathway. Binding of inhibitor to the regulatory subunits stabilizes the tense state or T state of the enzyme thereby preventing the reaction from taking place.

PLEASE REDRAW ALL FIGURES.First show the figure on top with its labels. Next show the left arrow appearing with the violet circles binding to the red circles as shown. Next show the right arrow appearing with blue circles binding to the yellow regions as shown.

Example for allosteric/feedback inhibition – Aspartate Transcarbamoylase

Enzyme

Stabilized R stateStabilized T state

CTP

CTP

CTP

CTP

CTP

PALA

PALA

PALA

SubstrateInhibitor

Catalytic subunits

Regulatory subunits


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Isoenzymes are homologous enzymes within a single organism that differ slightly in their amino acid sequence but catalyze the same reaction. These enzymes are mostly expressed in different tissues and have differing kinetic parameters such as substrate affinity (Km) and maximum velocity (Vmax). They may also have different regulator molecules that allow them to be differentially expressed and regulated depending on the requirement for that particular tissue.

First show the green & brown ‘isozymes A&B’ appearing. The arrows with labels must appear, flash a couple of times and then disappear. Next, the ‘substrate’ must appear, enter the ‘catalytic site’ as shown where it must change shape and then leave as the ‘product’. This must take place simultaneously for both A&B. The reaction taking place in B must be faster than A as depicted in animation. Once this happens, the text message shown below in red must appear. Next the violet pentagon must bind to the ‘regulatory site’ on top in B but not in A as depicted. This is followed by text message on top.

Isozyme forms

Tissue A – isozyme A

Tissue B – isozyme B

Different regulatory site

Similar catalytic site

Substrate

Product

Substrate

Product

Same reaction catalyzed – different kinetic parameters

such as Vmax, Km.

Different regulatory molecules


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Lactate dehydrogenase is an enzyme involved in anaerobic glucose metabolism that is present as two isozyme forms in human beings. The tetrameric heart enzyme , which requires an aerobic environment to function, has higher affinity for its substrate than the muscle enzyme. Despite having 75% sequence homology, they also differ in that high levels of pyruvate allosterically inhibit the heart enzyme but not the muscle form.

PLEASE REDRAW ALL FIGURES.First show the figure of heart and muscle along with the ‘LDH’ enzyme. Next show the reaction in the centre followed by the text ‘high affinity…’. Next show the blue pentagon binding to the pocket on the left but being unable to bind on the right followed by the text below as depicted.

Example for isozyme regulation – Lactate dehydrogenase

HEART ISOZYME – H4 MUSCLE ISOZYME – M4

Lactate

Pyruvate

High affinity for substrate

Low affinity for substrate

Not inhibited by pyruvate

Inhibited by pyruvate

LDH LDH


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Reversible covalent modification is another commonly employed enzyme regulatory strategy. The most widely observed modification is phosphorylation, which is carried out by various enzyme kinases with the help of ATP as a phosphoryl donor. Some enzymes are more active in their phosphorylated forms while others are less active in this form. Dephosphorylation is carried out by the phosphatase enzyme. Enzymes involved in glycogen metabolism are regulated by reversible phosphorylation.

First show reaction on the left appearing along with the ‘less active enzyme’. A red cross must appear over the reaction arrow. The curved arrows towards the right must then appear followed by the ‘more active enzyme’ on the right. When this appears, the reaction must take place as shown on right. Finally the curved arrows towards the left must appear as depicted.

Reversible covalent modification

PSubstrate

Product

Enzyme Enzyme

Substrate

Product

Less Active More Active

ATP ADP

Pi H2O

Kinase

Phosphatase


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Apart from phosphorylation, which most commonly takes place at serine, threonine and tyrosine residues, other reversible covalent modifications include adenylylation, uridylylation, methylation and ADP-ribosylation which modify different amino acid residues of the proteins.

The rows of the table must appear one at a time.

Reversible covalent modification

Type of covalent modification

Amino acid modified

Donating group

Phosphorylation Ser, Thr, Tyr His ATP, GTP

Adenylylation Tyr ATP

Uridylylation Tyr UTP

ADP-ribosylation Arg, Gln, Cys NAD

Methylation Glu S-adenosyl methionine


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Several enzymes exist in their inactive forms, known as zymogens, where they do not possess any catalytic activity. In order to become active, they need to be activated by hydrolysis of one of more peptide bonds by various protases. This removal of certain regulatory residues irreversibly converts the enzyme into its active form. Unlike reversible modification, the enzyme is degraded after completion of catalysis.

First show the reaction coming in as depicted followed by the ‘inactive’ enzyme’ appearing on the arrow followed by the red cross. Next show the pie shaped object ‘protease’ entering which must cleave the red portion and remove it. The red cross must then disappear and the label for enzyme must change as shown.


Substrate Product

Enzyme inactive form (zymogen)

Protease

Enzyme active form


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Several digestive enzymes as well as clotting factors are regulated by proteolytic activation. Chymotrypsin, a digestive enzyme that hydrolyzes proteins in the small intestine, exists in its zymogen form within membrane bound granules after synthesis in the acinar cells of the pancreas. The proteolytic enzyme trypsin converts it into its fully active form by cleavage of a peptide bond between arginine at position 15 and isoleucine at position 16. The resulting enzyme known as pi-chymotrypsin is acted upon by other such molecules to yield the completely active and stable alpha-chymotrypsin which consists of three chains linked by inter-chain disuphide bonds.

First show the ‘inactive chymotrypsinogen’ above followed by appearance of the reaction on the left with the red cross on it. Next show the ‘trypsin’ which must cleave the blue rod as shown to give ‘pi-chymotrypsin’. The green ‘trypsin’ must again cleave the blue chain to give the figure below of alpha-chymotrypsin followed by the reaction sequence on the right as depicted.

Substrate Product

Chymotrypsinogen(inactive)

Example for proteolytic activation – Chymotrypsin

1 245

1 24515 16 Pi-chymotrypsin

Pi-chymotrypsin

1 13 16 146 149 245A-chain B-chain

C-chain-chymotrypsin (active)

Substrate Product

Trypsin


Interactivity option 1:Step No:1

Interacativity Type Options Results

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Match the following enzymes with their appropriate regulatory strategies.

Glycogen phosphorylase

Chymotrypsin

Aspartate transcarbamoylase

Lactate dehydrogenase

Match the following.User must be allowed to drag the A, B, C, D into the dotted boxes shown on the right.

A.

B.

C.

D.

1. Isozyme forms

3. Allosteric inhibition

2. Covalent modification

4. Proteolytic activation

Enzyme name Regulatory mechanism

User must drag & drop the A, B, C, D provided in the left column into the dotted boxes present in the right column. Every time the user drags and matches it correctly, that box must then turn green. If user gets it wrong, then it must move back automatically into the left column. Correct answers are 1-D, 2-A, 3-B and 4-C.

Questionnaire1

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1. Which of the following amino acid residues is not part of the catalytic triad of the active site

of chymotrypsin?

Answers: a) His b) Asp c) Ser d) Pro

2. Which metal ion bound to the enzyme carbonic anhydrase is responsible for its activity?

Answers: a) Zn2+ b) Fe2+ c) Mg2+ d) Mn2+

3. The attachment of a metal ion to water generates which of the following molecules?

Answers: a) Strong electrophileb)Strong nucleophilec) Weak electrophile d) Weak

nucleophile

4. S-adenosyl methionine is the donor group for which type of reversible covalent

modification?

Answers: a) Phosphorylation b) Adenylylation c) Methylation d) UDP-ribosylation

Links for further reading

Books:

Biochemistry by Stryer et al., 5th & 6th edition

Biochemistry by A.L.Lehninger et al., 4th edition

Biochemistry by Voet & Voet, 3rd edition

Fundamentals of Enzymology by Price & Stevens, 3rd

edition

enzymes: catalytic strategies & regulatory mechanisms harini chandra binding of a substrate to...

Documents

enzymatic reaction

uncatalyzed reaction

transient covalent bond

covalent catalysispart

bond formation

enzyme aid

thick line

enzyme catalytic site