Review 3 Flashcards
Line-Weaver Burke plot Equation
1/V0 = (Km/Vmax)(1/[S]) + 1/Vmax
Competitive Inhibition
- E + S (Binds to E)
- Apparent increase in Km and does not affect Vmax
- Increased [S] can overcome inhibition
- Think of the graph
Uncompetitve inhibition
- ES (Binds to the transition state)
- Apparent increase in 1/Vmax but decrease in Vmax (Because always stuck in transition state and not product formation)
- Increased [S] will not do anything lol
- Think of the graph
Non-competitive Inhibition (Mixed)
- E + S (Can bind here) and ES (can bind here)
- Apparent increase in Km and in 1/Vmax but decrease in Vmax
- Increased [S] does not relieve anything
- Think of the graph
Km
Think (k-1 + k2)/k1 => (Loss of [ES]/Formation of [ES] and that denominator is the key to explaining everything
Positive Cooperativity
Substrate binding increases affinity for subsequent substrate (Affect Km)
Negative Cooperativity
Substrate binding decreases affinity for subsequent substrate (Affect Km)
Non-cooperative Binding
Substrate binding DOES NOT AFFECT affinity for subsequent substrate (Affect Km)
Allosteric Activator
Binding increases enzymatic activity. The affect the Km
Allosteric inhibitor
Binding decreases enzymatic activity (Affect Km)
Feedback Loop effects
- Positive increase the rate
2. Negative loops decrease the rate
Homotropic vs. Heterotropic
Former the regulating molecule is a substrate of the enzyme
Latter the regulating molecule is not the substrate of the enzyme
Small Post-translational modifications to Enzymes
- Methylation (-CH3)
2 Acetylation (CH3-C=O)
- Glycosylation
Zymogens
Inactive enzymes that require covalent modifications
Enzyme Inhibition
- Uncompetitive
- Competitive
- Non-competitive (Mixed)
- Suicide (covalent) inhibition
Cholesterol, Unsaturated, and Saturated Fats
Cholesterol - Increase fluidity at low temperatures and decreases fluidity at high temperature
Unsaturated - Decrease fluidity
Saturated - increase fluidity
Mixed Inhibition
- E + S (Can bind here) and ES (can bind here)
- Apparent increase in Km and in 1/Vmax but decrease in Vmax
- Increased [S] does not relieve anything
- Think of the graph.
- The difference between non-competitive and mixed inhibition is that mixed inhibition binds to either the enzyme or the enzyme-substrate complex but has different affinities for each
Coenzymes
These are ORGANIC non-protein molecules that bind to proteins and are required for their function
Cofactors
These are INORGANIC non-protein molecules that bind to proteins and are required for their function
Apoenzymes
These are enzymes without their cofactors
Holoenzymes
These are enzymes with their cofactors
Cosubstrate
They reversibly bind and transfer chemical group
Prosthetic group
The molecule covalently bound to an enzyme
Oxidoreductase
Oxidation-reduction. Transfer electrons of hydrogen ions (oxidase, reductase, dehydrogenase)
Transferase
Groups are transferred from one location to another (aminotransferase, kinase)
Kinase
Transfers a phosphate group from ATP or other high energy carrier to a substrate
Phosphorylase
Catalyzes the addition of a phosphate group from an inorganic phosphate to a substrate
Hydrolase
Hydrolysis reactions (ATPase, protease, nuclease, lipase)
Protease
Hydrolyses peptide bonds (trypsin, chymotrypsin, pepsin)
Phosphatase
Removes phosphate groups from a molecule
Lyase
Catalyzes the cleavage of a bond or the synthesis (synthase) of a bond without the addition of an outside molecule (like water). Often forms or breaks double bonds
Ligase
Catalyzes condensation reactions with the hydrolysis of high energy molecule. Usually
Cytoskeleton
Intracellular scaffolding anchored to cell membrane to provide support and organization
Matrix
Extracellular support the tissue of the body (tendons, ligaments, cartilage, basement membrane)
Elastin
Tri-helical fiber and makes up most of the extracellular matrix, important for
strength and flexibility. Major component of the bone
Collagen
Extracellular, stretches and recoils like a spring, restores original shape of tissue
Keratin
Intermediate filaments found in epithelial cells. Functions to waterproof and strengthen the tissue. Found in hair and nails
Actin
Intracellular, make up of microfilaments and the thin filaments in myofibrils. Most abundant protein in eukaryotic cells.
Tubulin
Intracellular, make up mictrotubules. Provide structure, chromosome separation in mitosis and intracellular transoirt with kinesin and dynein
Kinesin
Positive end. They align chromosomes during metaphase, brings vesicles to membrane (neurotransmitters release)
Dynein
Negative end. The sliding movement of cilia and flagella, takes vesicles away from membrane (neurotransmitters reuptake)
Binding Proteins
Bind and transport or sequester molecules (hemoglobin, calcium binding protein, transcription factors)
Call Adhesion Molecules
They can be found on the surface of most cells and aid in the binding of the cell to the extracellular
matrix of other cells. Integral Membrane Proteins
Cadherins
They are glycoproteins involved in calcium-dependent adhesion. They hold similar types of cells together
(epithelial)
Integrins
They span both membranes, bind to and communicate with extracellular matrix and cellular signaling. They promote cell division, apoptosis, white blood migration, clotting.
Selectins
They weakly bind to carbohydrate molecules that project from other cell surfaces. They are expressed on white blood cells. They play a role in host defense, inflammation, and white cell migration.
G-Protein Linked Receptor Mechanism
- Epinephrine arrives at the cell surface and binds to a specific G-protein linked receptor
- The cytoplasmic portion of the receptor activates G-proteins, causing GDP to dissociate and GTP to bind in its place
- The activated G-proteins diffuse through the membrane and activate adenylyl cyclase
- Adenylyl cyclase makes cAMP from ATP
- cAMP activates cAMP-dependent protein kinases (cAMP-dPK) in the cytoplasm.
- cAMP-dPK phosphorylates certain enzymes, with the end result being mobilization energy. For example, enzymes necessary for glycogen breakdown will be activated, while enzymes necessary for glycogen synthesis will be inactivated, cAMP-dPK phosphorylation.
G-Protein Linked receptor Function
G-protein-linkedreceptoractivatesmanyG-proteins,eachG-proteinactivatesmanyadenylyl cyclase enzymes, each adenylyl cyclase makes lots of cAMP from ATP, each cAMP activates many cAMP-dPK, and each cAMP-dPK phosphorylates many enzymes
G-Protein Linked receptor basic process
- Ligand binds receptor
- Receptor exchanges GDP to GTP
- G protein becomes activated
- G protein either activates or inactivates target protein
- G protein hydrolyses GTP to GDP
- G protein becomes inactivated
Native Page
- Separates by mass to charge ratio.
2. Useful to compare size of charged proteins with similar size.
SDS-PAGE
- Separates based on mass only
2. SDS a detergent that disrupts all noncovalent bonds and binds to protein giving it a negative charge.
Isoelectric focusing
- Separates based on pI
- Gel has a pH gradient ( acidic at the anode, basic at the cathode)
- Proteins that are positively charged will begin migrating toward the CATHODE and proteins that are negatively charged will begin migrating toward the ANODE.
- When the protein reaches the portion of gel where the pH = pI, the protein takes on a NEUTRAL charge and will STOP.
Chromatography
- The more similar the compound is to its surroundings (by polarity, charge, and so on), the MORE it will stick to its surrounding and SLOWER it will move.
- Chromatography is preferred over electrophoresis if LARGE amounts of protein are being separated.
Stationary Phase
Can be charged, have pores or special affinities.
Column Chromatography
Polar silica or alumina beads, the more polar the protein, the SLOWER it will move
Ion-Exchange Chromatography
- Beads coated with charged substances
- A positively charged column will attract and hold a negatively charged protein as it passes though the column, INCREASES its retention time
Cation Exchange
Bind positively charged molecules
Anion Exchange
Bind negatively charged molecules
Size-exclusion Chromatography
beads have tiny pores which allow small compounds to enter slowing them down, large proteins can’t fit into pores and therefore the LARGE proteins will elute first
Affinity Chromatography
- Columns to bind any protein of interest by creating a column with high affinity.
- Use receptors or substrate for the protein, antibodies
Protein Structure
X-ray Crystallography and NMR Spectroscopy
Amino Acid Composition
- Hydrolysis followed by chromatography
- Edmans degradation sequences by selectively removing amino acids of the proteins from the N-terminal
which can be analyzed by mass spectroscopy.
