Enzymes: Mechanisms Of Action and membrane function Flashcards Preview

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1

Enzymes

-catalyze reversible reactions
- the specificity of each reaction is established through the use of specific enzymes that catalyze each reaction.
- diverse mechanisms of catalysis have evolved to solve the unique problems of synthesis, degradation, transport, replication, motility, and communication
- study through kinetic analysis of their behaviors

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Why are enzymes over inorganic catalysts?

- greater reaction specificity: avoids side products
- milder reaction conditions: (37C, pH 7)
- higher reaction rates: in a biologically useful timeframe
- Capacity for regulation: control biological pathways
-

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Proteins (enzymes)

-oxireductase
-transferase
-hydroplane
-leases
-isomerases
-ligase

4

RNA enzymes (Ribozymes)

-ribosomes
-hammerhead, hepatitis delta virus RNA, group I intron ribozymes

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Oxireductases

- transfer of electrons

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Transferases

-group transfer reactions

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Hydrolases

-hydrolysis reactions (transfer of functional groups to water)

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Lyases

- cleavage of C-C, C-O, C-S, and C-N bonds by condensation reactions coupled to cleavage of ATP or similar cofactor

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Isomerases

-transfer of groups within molecules to yield isomeric forms

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Ligases

-Formation of C-C, C-S, C-O, and C-N bonds by condensation reactions coupled to cleavage of ATP or similar cofactor

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Enzyme Nomenclature

-some are still known by traditional names : pepsin, kinase, trypsin
- a numeric system of nomenclature
- first digit denotes class name
- the second digit denotes subclass
- third digit denotes the acceptor atom
- fourth digit denotes the acceptor

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Prosthetic group

- tightly and stably incorporated into a protein's structure by covalent or no covalent forces

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Cofactors

-bind in a transient, dissociable manner either to the enzyme or to a substrate such as ATP

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Coenzymes

- serve as group transfer as group transfer agents and transport many substrates from one point within the cell to another

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Apoenzyme

- enzyme without the factor

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Holoenzyme

-complete enzyme with prosthetic group

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Pyruvate dehydrogenase Complex

-Enzymes:
1) E1- pyruvate dehydrogenase
2) E2- dihydrolipoyl transacetylase
3) E3 - dihydrolipoyl dehydrogenase
-Coenzymes
1) TPP - bound to E1
2) Lipoate-covalently linked to E2
3) Coenzyme A - substrate for E2
4) FAD - bound to E3
5) NAD - substrate for E3 - reduced by FADH2

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FAD and FMN

- flavin nucleotides are tightly bound to flavoproteins

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NAD+ and NADP+

- contains vitamins B3 niacin
- involved in redox reactions, carrying electrons from one reaction to another. The coenzyme is found in two forms. NAD+ is an oxidizing agent - accepts electrons from other molecules and becomes reduced.
- Niacin deficiency causes pellagra

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Tetrahydrofolate

- carrier of one C group
- deficiency causes low birth weight infant and spina bifida

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Models of Enzyme-Substrate Interaction

- enzyme catalyzed reactions, a strong interaction is formed between a substrate and a cleft or pocket on the enzyme's surface that forms part of a region called the active site.
- Lock and Key
- Induced Fit

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Lock and Key

The enzyme dihydrofolate reductase with its substrate NADP+ , NADP+ binds to a pocket that is complementary to it in shape and ionic properties

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Induced fit

-conformational changes induced by binding of a substrate

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Catalysis by Proximity

- effective molarity concentration and orientation of substrate molecules in the active site of enzymes will enhance the rate of reactions. Eg. Substrate channeling by multi-enzyme complexes

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Acid-base Catalysis

- ionizable functional groups of amino acid side chains and prosthetic groups contribute to catalysis by acting as acids of bases

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Catalysis by Strain

- enzymes bind their substrates in an favorable conformation to weaken the bond that will undergo cleavage. Eg. Stickies model

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Covalent Catalysis

-formation of a covalent bond between the enzyme and one or more substrates to create a more reactive enzyme. Eg. Group transfer reactions

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AA in General in Acid-Base catalysis

- many organic reactions are promoted by proton donors or proton acceptors
- the active sites of some enzymes contain amino acid functional groups that can participate in the catalytic process as proton donors or acceptors

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Example of Acid Base catalysis

-enzymes of the aspartic protease family, which includes the digestive enzyme pepsin, the lysosomal cathepsins, and the protease produced by HIV share this common catalytic mechanism
- two conserved aspartic acid residues are involved in acid-base catalysis

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Example of Covalent Catalysis

- catalysis serine protease, chymotrypsin, involves prior formation of a covalent acyl-enzyme intermediate
- AA residues at the active sit, Asp 102-His 57- Ser 195, functions as a proton shuttle during the proteolysis

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Structure of Chymotrypsin

- protein consists of 3 polypeptide chains linked by S-S bonds
- active-site AA residues are groups together in the 3D structure

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Recombinant DNA technology and Site-Directed mutagenesis

- overproduction of recombinant proteins using recombinant DNA technology provides an important tool for studying enzyme structure and function
- Recombinant proteins can be readily purified by affinity chromatography in large quantities
- generation of mutant recombinant proteins by site-directed mutagenesis provides mechanistic insights

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Isozymes

- catalyze the same reaction but may differ in reaction rate, inhibition by various substances, electrophoretic mobility, or immunologic properties.
- some is oxygen may also enhance survival by providing a backup copy of an essential enzyme.
- their pattern in serum may indicate organ damage. Eg. Alkaline phosphatase, lactate dehydrogenase, creating kinase

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Detection of Enzymatic activities

- Enzyme assays that produce a chromagenic or fluorescent product are ideal for rapid analysis of multiple samples

1) NAD(P) - dependent dehydrogenases
-spectrophotomeric assays
-assay at saturating [S], so that v is proportional only to [E]
-coupled assays
2) Enzyme-linked immunosorbent assays

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NAD(P) dependent dehydrogenases

- coupled assays
- hexokinase assay coupled with glucose-6-phosphate dehydrogenase assay

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Detection of Lactate Dehydrogenase Activity

-coupled colorimetric assay to measure liver disease/damage in serum

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ELISA

1) coat surface with sample antigen
2) block unoccupied sites
3) incubate with primary antibody against specific angtigens
4) incubate with antibody-enzyme complex that binds primary antibody
5) add substrate

-used in detection of: Mycobacterium antibodies in TB, Rotavirus in feces, Hep B markers, Enterotoxin of E. Coli in feces, HIV antibodies in blood samples

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Membrane Function

1) Barrier
-plasma membrane separates cytoplasm from external environment
2) Compartmentalize
-organizes internal components into separate organelles with specialized functions

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Biological problems with membrane function

1) Communication - mediates communication with environment, transmembrane receptors transfer extracellular signals into the cell to influence cellular function
2) transport of molecules - selective permeability across membranes- regulates cellular composition- acquire nutrients, excretes wastes, maintain intracellular pH, ion concentration and water balance
3) Exchange of molecules with external environment and between organelles - membrane transporters, vesicular transport

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Permeability

-solubility in bilayer
- gradients
-selectivity depends on: solubility in lipid bilayer, specificity of transport proteins
-permeable molecules are readily soluble in lipid bilayer
-impermeable molecules have low solubility in the lipid bilayer

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Gradients

-concentration gradients affect the rate of diffusion
-for ions the rate of diffusion is also affected by electrical potential difference
- concentration gradient + electrical gradient = electrochemical gradient

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Passive Transport

1) Simple Diffusion - down concentration gradient
2) Facilitated Diffusion
-down concentration gradient
- protein mediated by: rate greater than expected based on solubility in lipid bilayer, selective - specific carriers for each solute, saturable - reaches maximal rate

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Active Transport

1) Primary
- pumps salutes up a concentration gradient
-requires hydrolysis of ATP: ion pumps (ATPases), ABC transporters
2) Secondary
- Coupled transport of two salutes provides the energy: symporters and antiporters

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Secondary Active Transport

- coupled transport:
- co-transport of two molecules
-uses energy from moving one solute down its gradient to drive movement of second solute up it its grand inept
- symporters and antiporters

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Transport Proteins

- transporters or carrier proteins
- pumps : ATPases
-Ion channels

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Characteristics of transporters of carrier proteins

- bind one or more salutes
- conformational change
- passive or secondary active transport

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How carrier proteins change conformation

1. Ligand binding site is exposed on one side of the membrane
2. Ligand binding changes the shape of the carrier so that the ligand binding site is exposed on the other side of the membrane and the ligand is released
3. Without the ligand bound the conformation returns to the original position so a new ligand can bind and the cycle repeats

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Characteristics of Ion Pumps

- ATPases: primary active transport, up concentration gradients, hydrolysis of ATP

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Types of Ion Pumps

1) P-type: autophosphorylate, multipass transmembrane proteins
2) V-type (Vacuolar): multi-subunit proton pumps
3) F-type: multi-subunit proton pumps
4) ABC transporters : ATP Binding Cassette

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The Na+ K+ Pump

- p-type
1. Na+ binds
2. Binding initiates phosphorylation by ATP and binding of Pi
3. induces a conformational change that releases Na+ extracellularly and
4. Exposes binding sites for extracellular K+
5. Binding of K+ induces release of Pi that allows the conformation to
6. change back, release K+ in the cytosol and
7. The transporter is ready for another cycle

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Na+ K+ ATPase Cycle

- cardiac glycosides oubain and digitalis inhibit the Na+/K+ ATPase and are used to treat cardiac failure
- slowing the Na+/K+ ATPase decreases the sodium grandient
- Ca++ transport out of the cell by the Na+/Ca++ antiporters is reduced
- higher intracellular Ca++ levels increase the force of contraction

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P-type ATPases

1) Ca++ ATPase
- plasma membrane and sarcoplasmic reticulum
- maintains low Ca++ concentration inside cell, which is important for signal transduction and muscle contraction
2) H+/K+ ATPase
- secretion of H+ from gastric cells
- Proton pump inhibitors block acid secretion (Prilosec, Prevacid)

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V-Type ATPases

1) H+ ATPase
- use energy from ATP hydrolysis to move H+ across membrane up a concentration gradient
- lysosomes: acidification to maintain optimal pH for hydrolysis
- some plasma membranes: osteoclasts(bone resorption) and Renal cells (acid base balance)

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Transporters

1) ABC transporters
- ATP Binding cassette, highly conserved
- transport many types of molecules:
- sugars, AA's, lipids, proteins, and peptides
- MDR: removes toxic compounds from cells, implications for treatment
- CFTR: Cl- transporter

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P-type ATPases disease and health

H+ ATPase - gastric ulcers
Na+/K+ ATPase - heart failure treatment

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ABC transporters in health and disease

- ABCR - macular degeneration
- MDR - drug resistance ( cancer, TB)
- CFTR - cystic fibrosis
- ABCA1- hypercholesterolemia

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V-type ATPases in health and disease

H+ ATPase- Bone resorption
H+ ATPase - lysosomes acidification

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F-type Proton Pump in health and disease

ATP synthesis in mitochondria

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CFTR mutations

- Class I - defective protein production
- Class 2 - defective processing
- Class 3 - defective regulation
- Class 4 - defective conduction

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Characteristics of Ion channels

1) transport ions
- rapid movement
- passive transport
2) Selective
- narrow channels restrict passage ( on size and charge)
3) Gated
- regulated opening to permit transport

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Electrochemical Gradients

- passive transport
- flow depends on gradients established by ion pumps:
- concentration gradient
- electrical gradient

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Ion Channels: Structure

- multi-subunit, multi-pass transmembrane proteins
- K+ channel is prototype: 4 subunits with 6 transmembrane domains

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Ion channel selectivity: Size

- K+ channels can distinguish between K+ and Na+ based on size
1) Opening in the K+ channel seen from the end
2) the opening is too small for either hydrated K+ or Na+ ions to pass
3) Carbonyl oxygen lines the channel
4) the oxygens are placed to exactly match those attached to K+, and H2O is displaced
5) the K+ is seen shedding its H2O for carbonyl O, passing through the filter, and picking up H2O on the other side
6) Hydrated Na+ doesn't match the spacing and is blocked

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Ion channel selectivity: Charge

- Acetylcholine receptor:
- cation channel
- negative charge on residues inside pore
- Na+ and K+ permeable
- Cl- impermeable

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Ion Channels: Gating

- most ion channels are gated
- opening and closing of the gate mechanism regulates entry of ions: mechanically gated, ligand gated, voltage gated
- Few are not gated: Leak channels

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Ligand - gated Channels

- converts chemical signal to electrical signal
- triggers an action potential by causing a local change in membrane polarization that stimulates the opening of voltage-gated channels
- binding of a specific ligand causes a conformational change and triggers opening ( can be extracellular or intracellular)
- removal of ligand closes channel

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Acetylcholine Receptor

1) Specificity
- selective for specific ion
- binds a specific ion
2) inhibitors
- Competitive - bungarotoxin, curare
- Non-competitive - valium

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Voltage-Gated

1) 3 conformational states determined by membrane potential (polarity charge)
- resting = closed
- activated = open
- inactivated = blocked by inactivation loop (gate is still open)
-return to rest = closed

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Leak Channels

- open all the time
- K+ channels
- K+ transported down its concentration gradient out of the cell
- increase inside negativity of the cell

70

Establishing the Electrochemical Gradient

- leak channel lets K+ ions out down the concentration gradient
- excess possible ions outside creates an electrical potential difference across the membrane
- the electrical potential opposes the efflux of K+ and will reach a point where it balances the concentration gradient - this is the equilibrium potential
- the Na+ K+ pump maintains the concentration gradients

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Resting Membrane Potential

- Electrical gradient with inside negative relative to outside
- Chemical gradient : Na+ and Cl- high outside, low inside. K+ high inside, low outside
- K+, Na+, Cl- contribute to the resting potential, but the influence of Na+ and Cl- are small
- resting potential is close to the equilibrium potential for K+