Part 3 espyr1 S33 ProteinsIII

Protein III

Enzyme Inhibition

- Irreversible inhibitor

o Substance that causes inhibition cannot be inhibited

o Involves formation or breaking of covalent bonds to or on enzyme - Reversible inhibitor

o Substance binds to enzyme, but can be released o Non-covalent interactions (weak)

Reversible inhibition

Competitive inhibition

- Inhibitor competes with the substrates

- Binds to the same active site as the substrate binds - Can be overcome by increasing [S]

�_�^{= � �} _[��]

�0⁼

� �� �

�� + [�]

� = 1 + ^�

��

The value of the α is the function that determine the affinity of enzyme (>>1) - Note that � �� remains unchanged

- Only change in slopes

Uncompetitive Inhibition

- Observed in multi-substrate enzymes only

- Inhibitor binds in the other active site of the ES complex to change the structure of enzyme, causes inhibition

- CANNOT be overcome by increasing [S]!

� =_�^{���� �}

�^+�′[�]

�^{′ = + �} _�

�′

- Note that the ratio _�^�

�� remains unchanged

Mixed Inhibition

- Mixed with both competitive and uncompetitive inhibition

�0⁼

� _�� �

�� + �^′ � - Affects both K^{m and Vmax}

- Non-competitive inhibition (Special type of Mixed inhibition) o _Km value will NOT change

Enzyme Regulation

- Methods of regulation

o Control of enzyme availability

 Control the rate of synthesis and degradation

 At transcription: induction and repression o Control of enzyme activity

 Through conformational and structural changes to influence the substrate-binding affinity

 Allosteric regulation, reversible covalent modification, Proteolytic activation

Allosteric Regulation

- Affected by other substances binding to allosteric site other than active site - Conformation altered will influence the affinity of substrate binding - Allosteric modulator/effector

o Positive modulator (Activator) o Negative modulator (Inhibitor) - If the modulator and substrate are

o Similar - Homotropic (Oxygen in haemoglobin) o Different - Heterotropic

- Regulatory step for multi-enzyme metabolic pathways usually catalyzed by allosteric enzyme - An effective system of inhibition: Feedback inhibition (End-product inhibition)

o The end-product inhibits the first step (committed step) of the pathway. o Effective in regulating when there is excess end product production - Allosteric Enzyme kinetics

o Sigmoid saturation curve o _K0.5 = [S] when V₀=¹

2^{Vma x}

o Note that when K0.5 increases, the velocity increases (Activator) and vice versa

o NO CHANGE IN Vma x^!

Cooperative Binding Models describing allosterism

- Symmetry (concerted) model - Sequential Model

Symmetry Model - Conformations

o R state (Relaxed) ^{– Active}

 Bind substrate tightly o T state (Taut or tight) – Inactive

 Bind substrate less tightly

- In the absence of substrate, most enzyme molecules are assumed in T-state. However, when substrate is present, the enzyme shifts from T-state to R-state simultaneously.

Sequential Model

- The changing conformation from T-state to R-state (or R to T) follows the induced fit theory

- When one subunit of enzyme changes from T to R, it will induce the conformation change of another subunits

- The process occurs sequentially, NOT simultaneously

Covalent Modification

Enzymes regulated by this method are known as inter-convertible enzymes because it can be converted to either to its active form or inactive form

Modifying groups:

- Phosphoryl, adenyl, uridylyl, adenosine Diphosphate ribosyl, methyl

Due to the covalent modification, another enzyme is required to activate the enzymes – converter enzymes

Phosphorylation

- Addition of phosphate group – Converted enzyme = Kinases - Removal of phosphate group – Converted enzyme =

Phosphatase

- Glycogen phsphorylase

o Catalsye the hydrolysis of the terminal glucose residue from non-reducing end of glycogen chain

o This enzyme is a dimer with two identical subunits o Phosphorylase a is formed when phosphate is added

on the Ser¹⁴ side chain

Proteolytic Activiation

Zymogen- Inactive precursor which can be irreversibly activated to an active enzyme by cleavage of covalent bonds

- Examples,

o Chymotrypsinogen  π-Chymotrypsin  α-Chymotrypsin (Stomach and pancreas) o Prothrombin  Thrombin (Blood Clotting)

o Fibrinogen  Fibrin (Blood clotting)

Cofactors

Non protein components that participate in enzymatic reactions and regenerated for further reactions

- _Types

o Metal ions

o Organic molecules (coenzymes)

 Many are vitamins or

metabolically related to vitamins (It’s good to eat Vitamins!) - Metal ions are Lewis acids – they can behave as

Lewis acids in acid-base catalysis - Most of the coenzymes involve in redox

reactions, which provide energy to organisms Transiently associated cofactors act as co-substrates (NAD⁺)

Tightly bound cofactors are called prosthetic groups (FAD) – Vitamin B12

- Holoenzyme

o Catalytically active enzyme-cofactor complex - Apoenzyme

o Catalytically inactive enzyme resulting from removal of cofactor Apoenzyme

(_{� ������)} ^{+ Cofactor↔}

Holoenzyme (_{������)}

Enzyme Nomenclature

- International Commission on Enzymes system (1956) - Each enzymes are assigned

o 4-digit classification number o Systematic name

Hexokinase

(ATP:glucose phosphotransferase)

EC2.7.1.1

Enzyme Commission

Class No.

Applications of Enzymes

Immobilised Enzymes

- Confine enzyme physically to make it localized in a certain region of space - Benefits:

o So that it can be saved and reuse again o Recover from reaction mixtures

o Product not contaminated with enzymes o Able to stop reaction rapidly

o Stabilisation of enzymes

o Protected from processes which may denature the enzyme - Methods to immobilize enzymes

o Carrier binding

o Cross-linking

 Link with bi- or multi-functional reagent with covalent reaction o Entrapment

 Trap enzymes in gel matrices, microcapsules, hollow-fibres, or ultrafiltration membranes

 Either in lattic-type or microcapsule type

 Disadvantage:

 If bound too tightly, the substrate are unable to go in to react with enzyme OR after reaction the products are unable to come out from the entrapment box

 If bound too loosely, the enzymes will slip through the trap - Applications:

o Glucose meter

 Enzymes immobilized on the glucose meter, if there is high glucose content, reaction will occur, there will be colour change. It will be detected by the meter

o Blood substitute

 Put haemoglobins in one capsule (immobilized protein)

 Artificial blood

Disease Markers

Enzymes are in similar forms (isoenzymes)

- Catalyse same reaction but they contain slightly different genes Track the change in the enzymatic reaction

- Lactate dehydrogenase

o Two different subunits : M and H chains Covalent binding

Physical absorption Electrostatic forces

Biospecific binding

Sensitive to pH change

Changes the structure, causes enzyme to be less active

- If a person has heart attack, H⁴ is abundant. Doctor can track the changes. - _M4 favors the forward reaction; H4 favors the reverse reaction