Biolchem Exam 1 Lecs 6-10

Lipids: Hydrophobic or hydrophilic, soluble or water-insoluble?

Are Lipids polymers?

Functional groups?

Lipids: Hydrophobic & water-insoluble

Not polymers

Lipids — Have many fully-reduced C (-CH2-), also have polar “head group” (amphipathic)

Many fully-reduced C (-CH2-), also have polar “head group” (amphipathic)

6 major classes of lipids

Functions: Energy storage, protective coating, cell membrane, signaling

Fatty acids

• In humans, branched/unbranched, # of carbons, double bonds in cis/trans config?

• Building blocks for what?

Carboxylic acid “fatty“ w/ long chain

• In humans, fatty acids are unbranched, have an even number of carbons (usually 12-24), and can have double bonds in cis

• Building blocks for most lipids (except sterols)

Fatty Acids Naming:

18:1(Δ⁹) meaning

Which carbon is ⍺?

ω-3

By length of carbon chain and location of double bonds

18 Carbons (length), 1 double bond, Δ⁹ position of double bond

C2 carbon is ⍺ (next to carboxyl carbon)

ω-3, double bond is 3C from the end

ω-3 & ω-6 polyunsaturated fatty acids (PUFAs) are ____ in the human diet

essential

Lipid, Fatty Acid Nomenclature

20 carbons, 5 double bonds, double bonds on C5, C8, C11, C14, C17

Longer chain, less/more soluble

More saturated (w/ H), less double bonds, less/more soluble

Less soluble

Less soluble (can pack tighter together)

Fatty acid (lipid) MP ↓ as chain length ____.

Fatty acid (lipid) MP ↓ as saturation ____.

Increasing surface area ____ dispersion forces between hydrophobic chains.

Saturated fatty acids packing

MP ↓ as chain length decreases and saturation decreases (less H, more double bonds)

Increased dispersion forces

Can pack more tightly, (more SA contact), w/ stronger dispersion forces & higher melting point

Remember unit of unsaturation (UU) is a double bond

Triacylglycerols (triglycerides):

• What type of lipid

• Charge?

Triacylglycerols (triglycerides): Simple lipids w/ 3 fatty acids esterified to 3 alcohol groups of glycerol

• Storage lipid

• Neutral

• Energy store and reduced carbons

• Saponification: Hydrolysis of lipids in basic conditions, make glycerol

Longest C chain, highest MP

Least double bonds, most tight packing, most LDFs, highest MP

Saponification

Hydrolysis of triacylglycerols (storage lipid) into carboxyl salt, produce soap & disrupts membrane

“Fats” solid at RT; have ____ fatty acids

“oils” liquid at RT; have more ____ fatty acids

Wax:

“Fats” solid at RT; saturated fatty acids

“oils” liquid at RT; unsaturated fatty acids

Wax: Esters of fatty acids and a long chain alcohol

Glycerophospholipid

Membrane lipids (amphipathic), glycerol esterified to 2 fatty acid chains & phosphate head

• Usually one saturated and one unsaturated fatty acid

Sphingolipid:

• Phosphosphingolipids:

• Glycosphingolipid:

Sphingolipid: Sphingosine backbone w/ long carbon chain, amide-linked saturated fatty acid, & head group.

• Phosphosphingolipids: Has phosphate-alcohol head group

  

• Glycosphingolipid: Has carbohydrate group

Sterol

Structure of sterols → 4-fused-ring (type of lipid), core rigid & planar.

Isoprenoid

5-carbon isoprene unit

Double bond on each end

Cholesterol, where is it usually found

Sterol, w/ isoprene tail

• Major sterol in animals’ membrane

• 1 OH group, precursor for other steroid lipids

Steroid Hormones

Hydrophobic signaling molecule, small, diffuses across cell membrane

• Cholesterol derivative

What are Cholesterol derivatives

Steroid Hormones, Vit D, Bile Acids & Salts

How to identify/group these

Steroid hormones, Vit D, and Bile Acids are cholesterol derivatives (4-fused-ring + isoprene tail)

vs.

Fat soluble vitamins are iso-prenoids

Eicosanoids (based on what)

• NSAIDS do what?

Based on arachidonate fatty acid (20:4)

• Local (paracrine) hormones

• Inflammation, fever, disease

NSAIDs inhibit eicosanoids (no prostaglandin & thromboxanes) production

Plasma vs. Endomembrane

Is plasma membrane stationary or fluid

Plasma membrane: External boundary of cell

Endomembrane: Internal compartments of cell

• Plasma membrane is fluid, self-repairing, & selectively permeable

Membrane lipids have ____ shape

• Micelle

Cylindrical, allows for bilayer

• Micelle: 1 layer, formed by single chain conical lipids (fatty acids). Hydrophobic inner core

Bilayer how long?

Impermeable to what?

Fluid Mosaic Model:

30 Angstrom

Impermeable to polar and charged molecules

Fluid Mosaic Model: Lipids free to move laterally in membrane (seen by fluorescent labeling)

Lipid movement in bilayer membrane

Lateral diffusion: Stay within leaflet (2D), FAST

Transverse diffusion: Move between leaflets

• Slow and needs to be catalyzed

• Asymmetric distribution

How is membrane fluidity defined?

Membrane fluidity: How easily lipids undergo lateral diffusion

• Temp dependent

• Membrane alternates between disordered liquid-state & ordered solid state (transition temp between)

What is membrane transition temp dependent on?

Composition

• Longer fatty acid chains → ordered state, ↑ transition temp

• More cis-double bonds → disordered state, ↓ transition temp

What regulates membrane fluidity

Cholesterol:

• ↑ Temp, unsat. fatty acids compact (↓ fluid)

• ↓ Temp, Sat fatty acids reduce packing (↑ fluid)

Less severe change than transition temp

Lipid Raft (Lipid Microdomain)

• Location

• How does cholesterol pack

• Is this thicker thicker/thinner than surrounding membrane?

Microdomains of sphingolipids & cholesterol (like a wrap of both)

• Outer leaflet

• Packs w/ long, saturated fatty acid chains of sphingolipids

• Thicker than surrounding membrane

• Stable in membrane

How are lipid rafts enriched?

w/ long helical segments, two long acyl chain modifications, or GPI-anchors

What are the 3 ways proteins associated with membrane?

Integral membrane proteins

Peripheral membrane proteins

Lipid-linked proteins

Integral membrane proteins

• How to remove them?

Fully embedded in the membrane

• Transmembrane domain (hydrophobic region in middle)

• Detergent to disrupt hydrophobic region, destroys the protein

Peripheral membrane proteins

• How to remove?

On surface of the membrane

• Since only interacting w/ polar heads, remove w/ salt

Lipid-linked proteins:

• How to remove?

• What can be the chain?

W/ long hydrocarbon chains covalently attached to lipid linking it to membrane

• Some detergent required to release lipid chain from membrane

• Chain: fatty acids, isoprenoids, or GPI-anchored

Integral membrane proteins (single & multi-pass) form what?

• Hydropathy plot?

Form α-helices or β-sheets within the membrane

• H bonding inside and shields polar peptide bond

• Hydropathy plot shows hydrophobic region in transmembrane protein

Top region have hydrophobic domain (or alpha helix)

Membrane fusion needs what?

Change in curvature of membrane

What changes lipid shape & membrane curvature (to allow for membrane fusion)?

Lysophospholipids & phospholipase (hydrolyze fatty acid)

What creates lysophospholipids? What impacts head groups?

(For changing lipid shape for membrane curvature)

Phospholipase A1 & A2 creates lysophospholipids

Phospholipase C & D Affects the head groups

Lipid bilayer ____ to molecules that are large, polar, and/or charged.

Impermeable

What passes through lipid bilayer w/o help?

• Hydration shell?

Nonpolar gas & small, uncharged slightly polar molecules pass by simple diffusion (NO IONS). (Larger require transporter)

• Lose & regain hydration shell as pass through membrane

Simple diffusion

Facilitated diffusion

Active transport

Secondary Active Transport

Passive vs. Active transport (which requires energy)?

Simple diffusion: Molecules move ↓ conc. grad w/o energy or transporter.

Facilitated diffusion: Passive transport, w/ membrane protein

• Non specific channel (porin), specific carrier protein, or aquaporin (osmosis)

Active transport: Also uses carrier protein moves molecules against conc. gradient, needs energy

• Energy source ATP or downhill transport of another molecule (secondary active transport)

Transport proteins - Non-specific example

Porins (size limited channels always open)

Gated ion channels

• Are they saturable?

Open and close at a specific event, for specific group of molecules (ligand binding, voltage)

• No

Transporters

For specific molecule (saturable)

Conformationally-gated (only open @ one side at time)

Affinity based on conformation

3 Types of Transporters

Uniporter: Moves molecule in 1 direction (passive)

Cotransporter: Moves 2 molecules simultaneously

• Symporter: Moves 2 molecules same direction (active)

• Antiporter: Moves 2 molecules opposite direction (active)

Glucose Transporter (GLUT1)

Passive uniporter, facilitative diffusion of glucose down conc. grad

• High rate of transport

• Saturable (as glucose conc increases, transport is saturated)

• Stereospecfic

P-type ATPases

• Ca2+-ATPase:

• Na+-K+-ATPase:

P-type ATPases: Active cation transporters use ATP hydrolysis to transport ions up a gradient (primary active transport)

Ca2+-ATPase: Transports 2 Ca2+ out for each cycle of ATP hydrolysis, maintains low cytosolic calcium conc.

Na+-K+-ATPase: 3 Na+ out, 2 K+ in for each cycle of ATP hydrolysis (essential for neuron action potentials)

Transport Coupling

Symporter Na (w/ conc. grad, favorable) & Glucose (against conc. grad, unfavorable) in

• Glucose absorption (from intestinal space to cells)

Unfavorable transport coupled to favorable transport

Make this card better

_{Lec 7} Catalysts & Example

Increase reaction rates w/o being consumed

• Faster reaction rates

• Milder reaction conditions (narrow temperature/pH range)

• Greater rxn specificity (avoid side products, stereospecificity)

Chymotrypsin 20 yrs uncatalyzed, half a sec catalyzed

Carbonic Anhydrase 5 sec uncatalyzed, .000001 sec catalyzed

• Usually globular proteins (enzymes), small % are RNA ribozymes

Enzymes Stereospecificity

Enzymes are chiral, rxns produce molecules w/ absolute stereospecificity (no racemic mix)

Geometric specificity

Enzymes vary in degree of geometric specificity, some select for one molecule, & some allow for larger range

Active Site

Enzyme-catalyzed reaction takes place in active site

• Has AA residues bind substrate and catalyze breaking/forming of bonds

• Bind through multiple, weak interactions; complementarity and flexibility depend on the residues in the active site

Lock and Key vs. Induced Fit

Lock & Key: Must be exact fit, very selective

Induced Fit: Binding of the substrate induces a conformational change in the enzyme’s active site that promotes association; substrate-induced conformation; more flexibility

Oxidoreductase:

Transferase:

Hydrolase:

Isomerase:

Ligase:

Lyase:

Oxidoreductase: Oxidation-reduction (OH to aldehyde)

Transferase: Complete transfer of functional group from one molecule to next

Hydrolase: Cleave bond (break bond) by adding water

Isomerase: Make structural isomer, transfer functional group within molecule

Ligase: Glue, join two molecules, coupled with ATP hydrolysis

Lyase: Form/break double bond w/o without hydrolysis or oxidation

Cofactors

• Metal ions

• Coenzymes

  • Cosubstrates

  • Prosthetic Groups

Ezymes chemically modified by rxn, then undo chemical change

Cofactor: Enhance enzyme activity

Metals: Cu²⁺, Zn²⁺, Mg²⁺, Ni²⁺, Mn²⁺, Fe²⁺, etc

Coenzymes: Complex organic or metalloorganic molecules

• Prosthetic Group: Permanently binds to enzyme, does not leave

• Cosubstrates: Cofactor that associates for rxn then dissociates

Increases chemical diversity beyond 20 AA

Humans cannot synthesize coenzymes: true or false?

Coenzymes are derivatives of ______

True, cannot synthesize essential coenzymes from scratch

B Vitamins (from diet)

For a reaction to occur, products G must be _____ than substrates G

As reaction proceeds, [P]/[S] increases until ΔG: ____

Lower (ΔG < 0)

As reaction proceeds, [P]/[S] increases until ΔG: 0 (chemical equilibrium)

ΔG°′

[P]eq/[S]eq , inherent property of rxn

Keq @ equilibrium for rxn

Biological standard free-energy change

@ stnd cond. (25°C, 1 atm, 1 M), pH 7.0 & water

Unfavorable reaction (ΔG°′ > 0), or near equilibrium reaction (ΔG°′ ≅ 0), can made favorable (ΔG < 0) by ____________________________.

A ___-fold increase in the equilibrium constant results in a 5.7 kJ/mol (1.36 kcal/mol) decrease in ____

changing the concentrations of substrate and product (Le Chatlier’s Principle)

A 10-fold increase in the equilibrium constant (Keq) results in a 5.7 kJ/mol (1.36 kcal/mol) decrease in ΔG°′

Do enzymes shift equilibrium?

Does ΔG indicate rxn energy feasibility or reasonable time scale

No, but arrive faster

ΔG is if a reaction is energetically feasible; regardless of reasonable time scale (rxn rate)

Enzymes accelerate attainment of _____, do not change rxn’s _____ and _____.

Enzymes decrease the _______

Enzymes accelerate attainment of equilibrium, do not change rxn’s ΔG and Keq.

Enzymes decrease the activation energy ΔG^t

Highest point of rxn is _____

Enzymes facilitate formation of _______

Energy to form transition state is _____

What determines rate of rxn?

How do enzymes increase rxn rate?

transition state (X^‡)

transition state (X^‡)

Activation barrier (Activation energy)

Activation barrier (Activation energy)

Increase rxn rate by decreasing ΔG^‡.

S to X^‡ to P

What is rate enhancement

What is ΔΔG^‡

What is the stabilization to rate increase ratio

How much can 1 H-bond increase rate by?

Fold change in rate from uncatalyzed to catalyzed rxn.;

ΔΔG^‡: Energy saved by using catalyst (vs. no catalyst), energetic stabilization of the transition state by the enzyme

Stabilization of 5.7 kJ/mol (1.36 kcal/mol) increases rate by 10-fold;

By > 100-fold

Enzymes bind to what the best?

Bind transition states best

Binding to X‡ (transition state) is the majority of ΔΔG‡ (energy saved)

Transition state analogs (look alikes) are potent ______. Explain why.

Planar analogs have _____ fold higher binding affinity than proline or tetrahedral analog

Inhibitors

Stable molecules resembling T.S. of rxn; bc enzymes binds best to T.S., molecules mimicking T.S. also bind tightly to enzyme and inhibit activity.

160 fold

What are the 4 catalytic stratgeies

Approximation and orientation

General acid-base catalysis

Covalent catalysis

Metal ion catalysis

Approximation and orientation (Catalytic Stratgey)

Brings 2 substrates into proximity and orients them for rxn

General acid-base catalysis (catalytic strat)

• General Base accepts H to stabilize _______

• General Acid donates H to stabilize _____

What groups act as general acids & bases?

Are they pH dependent?

General Base accepts H to stabilize forming double bond

General Acid donates H to stabilize - charge in T.S.

Must reform to do again

Ionizable AA side chains with PKA near 7

(General base must be deproted, Acid proted, otherwise activity lost: pH dependence)

Covalent Catalysis (catalytic strat)

Schiff Base

Active site’s reactive group forms covalent bond w/ substrate (Stable covalent intermediate); enzyme nuc attacks substrate electrophile.

Schiff base: Amine attacks carbonyl, easier to store e- density on N than C. Forms between enzyme amine & substrate carbonyl. “Electron sink” to stabilize negative charge

Enzyme forms stable covalent intermediate with substrate.

What type of intermediate is produced in covalent catalysis?

Stable covalent intermediate, follows diff rxn pathway than uncatalyzed rxn, schiff base acting as “electron sink“ to stabilize - charge in uncatalyzed rxn

Metal Ion Catalysis

Catalytic role, electrostatic stability, generate nuc by increasing the acidity of molecule, redox rxns, bind & orient substrate in active site

• Include Mg²⁺, Ca²⁺, Zn²⁺, Fe²⁺/³⁺, Co²⁺, Ni²⁺, Cu²⁺, Mn²⁺

• In alcohol dehydrogenase, Zn²⁺ stabilizes a negative charge in T.S.

• In carbonic anhydrase, Zn²⁺ activates water as nuc by making it more acidic & stabilizes - charge.

• 1/3 of all enzymes are metalloenzymes, but not all metal ions are catalytic; some are only structural