MCB EXAM 2 Lecture Notes

8/11/2019 MCB EXAM 2 Lecture Notes

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Lecture 8 (Chapter 11)

Lipids: water-insoluble molecules that are highly soluble in organic solvents

Amphipatic: Amphi (both), pathic (suffering/feeling), Having 2 sides with opposite

properties. Having polar and non-polar end.

Philic:(love) so amphiphilic loves bothFive Classes of Lipids:

Fatty Acids(free fatty acids/nonesterified): simplest type of lipid that vary in

hydrocarbon chain length. Main source of fuel and major building blocks of

membranes. Can be saturated or unsaturated.

Triacylglycerol:storage form of fatty acids and major source of glycerol.

Phospholipids:main component of membranes. Consists of fatty acids attached to a

scaffold that bears charged phosphoryl group, creating a macromolecule with a

polar head and nonpolar tail.

Glycolipids: lipids that are bound to carbohydrates. important membrane

constituents

Steroids:polycyclic hydrocarbons with a variety of functions. Function as

hormones that control a variety of physiological functions. Most common is

cholesterol (vital membrane component)

Fatty Acids: amphipathic molecule that possesses both polar and nonpolargroups.

Carboxyl group is the head that is polar, hydrophilic (pKa ~ 2) is ionized at pH 7.

Tail: nonpolar, hydrophobic

When glucoses (partially oxidized) and fatty acids are broken down, they areconverted into CO2 and H2O. Fats yield more energy than carbohydrates when

undergoing combustion to carbon dioxide and water.

Fatty acids can have saturated or unsaturatedacyl chains. Saturated: only single

bonds. Unsaturated:has double bonds in hydrocarbon tail

Nomenclature/Notation for fatty acidsA:B, A is the number of carbons and B is

the number of double bonds. C=O bond should not be included when asked for # of

double bonds.

*do not have to remember common names of fatty acids

Carbonyl carbon is carbon #1.

A:B(cis-^1,2,3,4) 1: is the first carbon that a double bond starts at.


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Also there is(omega) nomenclature: reversed numbering (last carbon in chain is

first) Fatty acid is named after first double bond that appears(omega)-# fatty

acid. Last carbon in chain is called the omega carbon atom.

Carbon atoms 2 and 3 are often referred to as alpha and beta respectively.

Most of the common fatty acids found in nature have an even number of carbonatoms and no more than 4 double bonds (typically between 14 and 24 carbons) 16

and 18 are most common.

anoicno double bond

enoicdouble bond present

Just like amino acids, fatty acids are ionized at physiological pH so preferable to

refer to their carboxylate from vs carboxylic.

Mono vs. poly fatty acids: based on number of double bonds

There are cis and trans unsaturated fatty acids.

Trans fatty acids: trans fat, shape is not changed (partial hydrogenation of fatty

acids, processed) trans double bonds are less prone to oxidation

Cis fatty acids:theres a kink, linear structure/shape is somewhat changed

Most naturally occurring unsaturated fatty acids almost always are the cis

configuration.

Trans/ cis, saturated/unsaturated affects melting temperature.

Unsaturated fatty acids have lower melting points than those of saturated fatty acidsof the same length.

Lower melting T correlates with more fluidity.

Van Der waals interactions occur between hydrocarbon tail in fatty acids More van

der waal interactions present lead to a higher amount of heat which means a higher

melting temp and more energy is needed to separate the van der waal interactions.

The more kinks (cis double bonds) the easier it is to separate van der waal bonds,

and the lower the melting temperature. So cis double bonds lower melting

temperature.

Trans double bonds look like saturated fatty acids so its melting temp is still higherthan cis.

Chain length also affects the melting point. Short chain length and cis unsaturation

enhance the fluidity of fatty acids and lowers melting temp.

Unsaturated, cis fatty acids are known to be good fats because we cannot

synthesize them.

Some fatty acids are essential components of our diet (omega-3 and omega-6)


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Concentration of free fatty acids in cells or the blood is low because free fatty acids

are strong acids. This would disrupt the pH balance of cells.

Fatty acids required for energy generation are stored as triacylglycerols(3fatty acid chains bonded to a glycerol, each carbon of the glycerol is esterified). there

are simple triacylclycerol (same fatty acid chains) and mixed (different fatty acid

chains)

Common soaps are the sodium or potassium salts of fatty acids generated by

treating triacylglycerols with strong bases.

A gram of nearly anhydrous fat stores more than six times as much energy as a gram

of hydrated glycogen.

Most natural plant and animal fat are triacylglycerols

Glycogen and glucose stores can maintain biological activity for about 18-24 hours.

Tg stored allows survival for several weeks

Triacylclycerol is a good fuel:

1)

fatty acids are richer in energy (more reduced) than carbohydrates.

Complete oxidation of Tg yields 38 kJ energy/ig (more than 17kJ/g of protein

or carbohydrate)

2)

tg can be stored more efficiently. In a neutral way that does not affect cellular

processes

3)

tg aggregates are inert and there is no risk of undesired chemical reactions

with other cellular components

4)

provides enough stored energy to last weeks

Soap is made up of sodium and potassium salts of long chain fatty acids

Soap is an emulsifying agent. Aqueous liquid with organic solvent and

hyrophobic/hydrophilic parts.

Adipose cellsare specialized for the synthesis and storage of triacylglyceros and for

their mobilization into fuel molecules that are transported to other tissues by the

blood. Adipose tissue also serves as a thermal insulator.

3 common types of membrane lipids:

1) Phospholipids:has to have a phosphate group

2)

Glycolipids: has to have glucose3) Cholesterol:does not contain fatty acids

Lipids: can be fatty acidsor isoprenoids/steroids(cholesterol)

Fatty acids phosphoglyerides/glycerophospholipids,

trigylcerides/triacylglycerides. Sphingolipids


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Phospholipids are abundant in all membranes. Composed of one or more fatty acid

attached to a platform attached to a phosphate which is in turn attached to an

alcohol. (Backbone is usually a sphingosine or glycerol)

Phospholipids come from glycerides and sphingolipids. They have hydrophilic and

hydrophobic components.

Phosphoglyceride has a glycerol as a backbone. Only first 2 carbons of

phosphorglyceride are esterified to fatty acid chains. 3 carbon is bonded to

phosphate and alcohol. Simplest phosphoglyceride is phosphatidate.

Phosphotidates: only small amounts present in membranes. But is a key

intermediate in the biosynthesis of other phosphoglycerides and triacylglycerols.

Made up of Phosphotidyl- ethanolamines, serines, cholines, inositols and

cardiolipinare the alcohols that can be found bonded to phosphate.

Sphingolipids:have a sphingosine backbone instead of glycerol. Have ahydrophobic tail. (sphingomyelin is common)

Sphingosine is a amino alcohol that contains a long unsaturated hydrocarbon chain.

Sphingomyelins: phospholipids found in membranes. Sphingosine backbone. It can

bephosphoryl(choline/ethanolamine).Fatty acid attached via an amide linkage

to the sphingosine backbone

Sphingomyelinphospholipids

Glycolipids are sugar-containing lipids. Derived from sphingosine. Differ from

sphingomyelin because it is linked to one or more sugars instead of

phosphorylcholine.Simplest glycolipid is a cerebroside. More complex are gangliosides.

Cerebrosides/gangliosides glycosphingolipids (always have a sphingosine

backbone attached at the 1-OH group of sphingosine)

One sugar: cerebroside

Two or more sugars: ganglioside

In ceramide, fatty acid is attached via an amide to sphingosine backbone

Ceramidessphingomyelin, cerebrosides, gangliosides

Steroids:lipids that have a variety of roles. Made up of cyclohexane rings and

cyclopentane. Powerful hormones, facilitate the digestion of lipids in the diet, are

key membrane constituents.

Steroid nucleus: made up of 3 cyclohexaness and 1 cyclopentane


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Cholesterol:made of isoprene units. Most common steroid. Important in

maintaining membrane fluidity. Absent from prokaryotes but found in all animal

membranes. Constitutes almost 25% membrane lipids but absent from some

intracellular membranes. Free cholesterol does not exist outside of membranes

(esterified to fatty acid). Cholesterol disrupts the tight packing of fatty acid chains

Cholesterol derivatives:

1)

5 families of steroid hormones: androgens, estrogens, progestins,

glucocorticoids, mineralocorticoids

2)

bile acids: assist in absorption of dietary lipids in the intestine

3) vitamin D ( calcium absorption)

Membrane lipids are amphipathic (hydrophilic and hydrophobic)

3 types of lipid anchors(proteins covalently bond to lipids to localize the protein

to the cell membrane)

1) palmitoyl group attached to a cysteine residue by a thioester bond

2) Farnesyl group attached to a cysteine residue at the carboxyl terminus

3) Glycosylphosphatidylinotisol (GPI) anchor: glycolipid structure

attached to the carboxyl terminus

GPI anchor is always found on the outside of the cells

Hutchinson-Gilford Progeria Syndrome (HGPS): caused by inappropriate

fanesylation. Mutation in the gene for the protein lamin.

Lamin processing is different in HGPS patients (progerin instead of lamin A)

Eukaryotic membranes:membranes serve as boundaries that maintain division of

labor in cell. Membranes are actively involved in cellular processes. Permeability

barriers

Membranes are bilayer. Phospholipis and glycolipids form lipid bilayer in aqueous

solutions. Polar head groups + hydrophobic tails

Membranes formation:lipids form ordered structures spontaneously in water

Driving force behind amphipathic lipids to form ordered structures in aqueous

solutions is: waters tendency to form H-bonds and share in polar interactions.Hyrophobic affectpromotes self association of lipids in water to maximize water

entropy

Membrane composition reflects its function: membrane fluidity is controlled by

fatty acid composition and cholesterol content.


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Below melting temperature, membrane is rigid, solid (ordered structure) because of

van der waal interactions. Above melting temperature, membrane gains fluidity.

In animals, membranes have cholesterol. Cholesterol disrupts the van der waals

interactions in the tight packing of the fatty acid chains, which makes the membrane

more fluid. At higher temperatures, cholesterol is very rigid which limits ability ofchains to move around. Limits the range of fluidity/solidity of membranes.

Membranes are 2-dimensional solutions of oriented globular proteins and lipids.

Properties and characteristics:

1)

Sheet like structures

2) Composed of lipids and proteins

3)

Membrane lipids are small amphipathic molecules

4)

Proteins serve to mitigate the impermeability of membranes5) Membranes are noncovalent assemblies. Hydrophobic affect

6)

Membranes are asymmetric

7) Membranes are fluid structures

Layer that faces cytoplasmic side: inner leaflet

Layer that faces extracellular space: outer leaflet.Anything with sugars are on the

outer leaflet (glycolipids)

Transverse Asymmetry:between leaflets.

Lateral Heterogeneity:(along membrane) Different regions have different

concentrations of lipids/proteins. Not evenly distributed.

Certain enzymes (called flipaes) flip lipids back to where they belong.

Lateral diffusion: lipids diffuse through the membranes very rapidly (2uM/sec)

Transverse diffusion (flip-flopping) is very slow

The fluid mosaic Model:

Cholesterol, oligosaccharide side chain(OUT), peripheral protein(IN), integral

proteins(IN), phospholipid membrane, glycolipid(OUT).

Membranes composed almost entirely of lipids are suitable for insulation becausehydrophobic components do not conduct currents well. The plasma or exterior

membranes conduct the traffic of molecules into and out of the cells. Protein content

of plasma membranes is around 50%. Energy transduction membranes

(mitochondria/chloroplast) contain higher amount of protein, around 75%.

Heterokaryons: consists of 2 different cells (human and mouse cells) Then the cells

are fused (proteins on the cell surface are detected by specific antibodies), it then


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becomes one big heterokaryon and after some time (40 min) the human and mouse

proteins are intermixed in the plasma membrane.

Fluorescence Recovery after photobleaching (FRAP):

Starts with one cell then a laser beam is pointed at specific area in cell which

bleaches the fluorescence. After bleaching certain region, green fluorescence proteinwill denature (unfold) so the protein is no longer able to fluoresce. See how long it

takes to recover/ gain fluorescence. GRAPH on slide 11. Some proteins move faster

than others so you can observe this with FRAP. USES lateral diffusion.

Different proteins associated with membrane processes:

Transporters

Anchors

Receptors

Enzymes

Membrane Proteins are classified as Integral or Peripheral:

Integral membrane proteins enter hydrophobic environments (can enter both

leaflets or just one) can be released only when physically disrupted.

Peripheral proteins: interact with the outside of the head (hydrophilic). They can be

bound to the integral proteins. Primarily bound to the head groups of lipids by

electrostatic and hydrogen-bond interactions.

Differentiating is based on how easy it is to remove protein from membrane

(peripheral does not need treatment, easier) Integral protein needs full dissocation

(harder to remove.)

Proteins in membranes are associated with alpha helices and beta

strands/pleated sheets. Portions that are in contact with nonpolar core of the

lipid bilayers are dominated by alpha helices or beta sheets because these second

order structures neutralize the polarity of the peptide backbone through h-bond

formation.

Integral membrane proteins can embed part of the protein onto the membrane.

Dimers form hydrophobic channel(hydrophobic amino acid side chains)

Prostaglandin H2 synthase-1(involved in pain from injury)

The channels formed create a hydrophobic environment. Serine 530 (Ser 530) is aregion within hydrophobic channel that aspirin can modify and inhibit

cyclooxygenase activity by obstructing the channel. So aspirin reduces

prostaglandin and pain.

Membrane-spanning alpha-helices are a common structural feature of integral

membrane proteins.

Also beta strands can be used to form a pore in the membrane.


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Aspirin inhibits cyclooxygenase activity by obstructing the channel.

Single transmembrane segment: e.g. glycophorin A is an integral protein in the

membranes of red blood cells.

Hydropathy plots: x axis is residue number, y axis: hydropathy index. Hydrophilic:

have negative numbers. Hydrophobic have positive numbers (single

transmembrane protein).

Transport across membranes:

Simple diffusion

Facilitated diffusion (passive transport)

Simple and facilitated both have transport from high to low concentrations

(thermodynamically favored direction, no energy input required) e.g pores,

channels, carriers.

Active transport: moves in thermodynamic unflavorable direction (low to high

concentrations) needs energy to drive the process. E.g. pumps

[C2]-[C1] is the concentration gradient.

High concentration has lower entropy, more ordered.

Low concentration has higher entropy

Molecules spontaneously move from a region of higher concentration to one of

lower concentration.

Lipophilic molecules can pass through the membrane because the dissolve in the

lipid bilayer.Na+ cannot enter because it is a charged ion.

In charged molecules, charge differences across the membrane must be taken into

account.

A nerve impulse, or action potential, is an electrical signal produced by the flow of

ions across the plasma membrane of a neuron. In par-ticular, Na+ transiently flows

into the cell and K+ flows out.

Inside of membrane cells are high in potassium concentration (lower on the

outside). Because of the difference in charge, potassium cannot be balanced equally.

Extracellular: positively charged

Intracellular: negative

Simple diffusion: movement until concentration inside and outside is the same.

Thermodynamically favorable


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Lipid bilayet (nonpolar,hydrophobic) are more impermeable(less permeable) to

ions and most polar molecules. Based on a log scale.

Ability of small molecules to cross a membrane is a function of its hydrophobicity.

Facilitated diffusion using carriers(from high to low concentrations) does not

require energy. Requires a carrier protein. Transmembrane protein carriers

changes shape to facilitate entry and exit of some nutrients.

Facilitated passive diffusion (uses pores/channels) this is regulated because it can

be closed or open. 1) ligand activated, 2) voltage activated. Channels display a

measurable affinity and specificity for the transported solute. Channels are gated

Ligand activated channels: ligand is a signaling molecule. Ions can go in when gate

opens. When ligand dissociates, gate closes again.

Voltage activated channels:action potential: at rest, negative charges are inside.

Once event triggers signaling, action potential enters and negative charges change to

positive (change in voltage opens channels and sodium flows in). Potassium

channels also open and allow K+ to flow out of cell.

Channels have affinity filters that only allow specific ions to flow in or out.

Pottasium channel:higher K+ concentration has larger diameter (10 Angstroms)

where K and Na can go in and lower K+ concentration has smaller diameter (3

angstroms) where only K+ can go in. Hydration shell needs to be lost to allow K+

ions to enter the narrow part of channel.

Thermodynamically unfavorable to lose hydration shells is made up by the carbonyl

oxygens from selective filters.

Selective filtersare carbonyl oxygens. Interactions can only take place with 2

carbonyls at a time. (slide 40)

K+ channel transport continued: K+ repels which pushes a K+ out of the cell.

Facilitated vs Simple Diffusion:

Both transport molecules down their concentration gradient.

Simple diffusion has linear form between [S] and velocity.

Facilitated diffusion has a curved first increasing then slope decreases. (curved) this

is because of the saturation of carriers (when carriers are gone, process is slower)


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Apoenzyme:enzyme without its cofactor

Holoenzyme:complete, catalytically active enzyme.

Cofactors:1) small organic molecules (derived from vitamins called coenzymes)

2) metals

Prosthetic (helper) groups: tightly bound coenzymes

Coenzymesdiffer from substrates because the are used by a variety of enzymes.

Different enzymes that use the same coenzyme usually carry out similar chemical

transformations.

Free energy (G):thermodynamic property that is a measure of useful energy, or

energy capable of doing work.

The difference in energy between products and reactants determines whether a

reaction will take place spontaneously.

Free energy required to initiate the conversion of reactants into productsdetermines the rate of reaction.

A reaction can take place if G is negative. spontaneously or without input of

energy. Also called exergonic reaction

An input of free energy is required when G is positive. (not spontaneous) also called

an endergonic reaction.

A system at equilibrium means G is zero and there is no net change in concentrations

of products and reactants.

The G is independent of path or molecular mechanism

G provides no information about rate of reaction.

The standard state is defined as having pH = 7. So when H+ is a reactant, its

concentration is 1 M. (concentration of water is also 1 in this state)

An enzyme cannot alter the laws of thermodynamics and cannot alter the equilibrium of a

chemical reaction.

Enzymes accelerate the attainment of equilibria but do not shift the position. The

equilibrium position is a fxn of only free-energy difference between reactants and

products.

Transition state:(double dagger) fleeting molecular structure that is no longer the

substrate but not yet the product. Least stable and most-seldom occurring species along

reaction pathway because it has the highest free energy.


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Free energy of activation or Activation energyis the difference between transition

state and substrate.

Enzymes facilitate the formation of the transition state. Catalysis is based on stabilization

of transition state.

Enzymes bring together substrates in enzyme-substrate (ES) complexes. The substrateor substrates are bound to specific region of the enzyme called the active site.

The interaction of the enzyme and substrate at the active site promotes the formation of

the transition state. Active site is the region of the enzyme that most directly lowers the

G (transition state/double dagger)

Common features of active sites:Active site is a 3-d cleft or crevice formed by groups that come from different parts of

amino acid sequence.

Active site takes up a small part of the total volume of enzyme. Most of the amino acidresidues in an enzyme are not in contact with the substrate. Extra amino acids serve as a

scaffold to create 3-d structure and constitute regulatory sites (sites of interaction withother proteins or channels to bring the substrates to the active sites).

Active sites are unique microenvironments: water is usually excluded from active siteunless it is a reactant. Nonpolar microenvironment enhances the binding of substrates as

well as catalysis.

Substrates are bound to enzymes by multiple weak attractions: noncovalent bonds

mediated by the hydrophobic effect (van der waals, electrostatic, and hydrogen bonds).

To bind as strongly as possible, enzyme and substrate should have complementarystructures.

Specificity of binding depends on the precisely defined arrangement of atoms in an active

site. (need matching shapes to fit together)

Lock and key analogy. Induced fit: active site assumes shape that is complementary tothat of the substrate only after the substrate has been bound.

Binding energy:free energy released from the binding of substrate and enzyme.Full complement of interactions between enzyme and substrate is formed only when the

substrate is in the transition state. (Maximal binding energy is at transition state).

Transition-state analogy:compounds that resemble the transition state of a reaction butare not capable of being acted on by the enzyme.

The inhibitor power of transition state underscores the essence of catalysis: selective

binding of the transition state.


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Catalytic antibodies or abzymes: antibodies generated that recognize the transition

states of certain reactions.

Lock and key model is harder to bend (tightly bound)

Active site is complementary to transition state.Enzyme stabilizes the transition state.

Enzyme inhibitors work best when it looks like a transition state, not a substrate.

Proline racemase L-Prolinetransition stateD-Proline

Transition-state analog (pyrrole 2-carboxylic acid)

Enzyme Kinetics Lecture 11 (Chapter 7)Allosteric enzymes: prevent chaos and allows for the efficient integration of

metabolism.

Kinetics is the study of rates of chemical reactions.

2 Types of Enzymes:

Michaelis-Menten: their activity is a function of substrate concentration

Allosteric: regulated, information sensors

Enzymes exert kinetic control over thermodynamic pontentiality (forward or

backward)

Kinetics: First Order Reactions: velocity is directly proportional to reactant

concentrations (units s-1)

SP

V = -d[S]/dt = d[P]/dt

Two reactants second-order reactions or bimolecular reactions

2AP

or

A + BP

V = k[A]2or V = k[A][B]

Some cases have zero order reactions which means rate is independent ofconcentrations.

Irreversible:

V = k[S] (V = velocity) depends on concentration of substrate and rate constant k


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Why does the first order graph (product vs time) eventually level off?running out

of substrate (analogy to popcorn)

Linear relationship between reactant concentration [S] and initial velocity Vo. Slope

= k (time-1)

Reversible reactions: At equilibrium k1[S] = k-1[P]

Biomolecular reactions where S2 is in excess of S1, reaction becomes pseudo first

order. S1 + S2P1 + P2 (graph for product vs time looks like first order reaction)

Linear curve between V vs [S1] in pseudo first-order. [S2] >> [S1]

Effect of enzyme concentration on initial velocity at a fixed saturating substrate

concentration ([S]>>>[E])

Pseudo first-order reaction

Kinetic of Enzyme catalyzed reactions (keeping [Enzyme] constant but increasing[substrate]Initially first-order reaction with respect to S but levels off to zero

order reaction with respect to S (Vo vs [S])

Zero order reaction: adding more substrate no longer affects velocity because of

saturation. (Every enzyme has gone to substrate) Vo reaches V max.

E + SESE + P (all reversible)

K1/K-1, K2/K-2

K = rate constant

Enzymes are neither reactants nor products

Michealis-Menten (MM) equation: describes variation of Enzyme Activity as a

function of Substrate Concentration

E + SESE + P

When ES complex is formed, it can either dissociate to E + S (k-1)or proceed to form

product (k2)

MM Equation: V0= Vmax[S]/ Km+ [S]

V0= initial velocity

Vmax= Maximal velocity

Km = Michaelis Constant is unique to each enzyme and independent of enzyme

concentration.

Km= (K2 + K-1)/K1

[S] = substrate concentration


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Affinity of E for S is inversely proportional to Km

Vmax = K2[Et] (directly dependent on enzyme concentration)

V0 = Vmax[S]/(Km + [S])

When [S] is much less than KmVo = (Vmax/Km)[S]When [S] is very largeVo = Vmax

[S] = Km when Vo = Vmax/2

V0 = Vmax[S]/2[S]

To reach Vmax, need infinite amount of substrate concentration [S]

Physiological consequences of Km

Mitochondrial aldehyde dehydrogenase has low km but inactive in certain

population. (having more of this allows you to control the toxic effects of drinking

too much)

Cytoplasmic aldehyde dehydrogenase has high Km.

Low Km mitochondrial form vs high km cytoplasmic form.High kmhigh rate of catalysis only at very high concentrations of acetaldehyde

less converted to acetate and excess acetaldehyde escapes into the blood.

Evidence suggests that the Km value is approximately the substrate

concentration of the enzyme in vivo.

A double reciprocalthe Lineweaver-Burk plot

Y = a x +b

1/Vo = Km/Vmax * 1/S + 1/Vmax

slope: Km/Vmax

Y axis: 1/VoX-axis: 1/[S]

For most enzymes: Km lies between 10^-1 and 10^-7 (depends on pH, temperature,

ionic strength)

Turnover Number= number of substrate molecules that an enzyme can convert

into product per unit time when the enzyme is fully saturated with substrate.

Turnover number = k2 or Catalytic constant (kcat) Most turnover numbers are

between 1 to 10^4 per second

When the enzyme is saturated with substrate K2 = Kcat

Kcat = Vmax/[Et]

Measures the effectiveness of an ezyme. Usually the limiting step.

Catalytic Efficiency: if [S]


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Most reactions in biological systems are Bisubstrate Reactions:

A + BP + Q (reversible)

Sequential reactions:random vs ordered: all substrates must bind to the enzyme

before any product is released. A ternary complex consisting of the enzyme and bothsubstrates forms.

Conversion of pyruvate to lactate by lactate dehydrogenase

Double displacement/ping-pong reactions:one or more products are released

before all substrates bind the enzyme. Existence of a substituted enzyme

intermediate in which the enzyme is temporarily modified.

Allosteric enzymesare catalysts and information sensors. Allosteric enzymescontrol the flux of biochemical reactions in metabolic pathways.

Most enzymes in the cell are Michaelis-Menten enzymes.

Catalysis is not enough in functioning of a cell. Vast array of reaction pathways also

need to be regulated.

Allosteric enzymes: regulate the flux of biochemical through metabolic pathways.

Key features of allosteric enzymes:regulation of catalytic activity by

environmental signals, including the final product of the metabolic pathway

regulated by the enzyme. Kinetics for allosteric enzymes are more complex than M-M and they have quartenary structures with multiple active sites.

Feedback inhibition:common means of biochemical regulation. Bears no

structural resemblance to the substrate or product of the enzyme that they inhibit.

Feedback inhibitors do not bind at the active site but rather at a distinct regulatory

site on allosteric enzyme. (allos : other, stereos: structure)

Allosteric enzymes always catalyze the committed step of metabolic pathways.

They can recognize inhibitor molecules and stimulatory molecules.

Allosteric enzymes differ from M-M. Graph is sigmoidal with sharp increase of oin the middle of the curve. More sensitive to changes in [S] near Km which allows for

more sensitive control of reaction velocity.

Allosteric enzymes are quaternary structures.

Concerted/MWC model:have multiple active sites on different polypeptide chains.

Can exist in R (relaxed) or T (tense) state. R is active conformation which catalyzes

reactions. T is less active. In the absence of substrate or signal molecules, R and T


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are in equilibrium. T/R is the allosteric constant Lo. Concerted model requires that

all of the subunits or active sites must be in the same state (all R or all T) This is

called the symmetry rule. S binds more readily to the R form than to the T form.

The binding of substrate disrupts the TR (reversible) equilibrium in favor of R.

This is called cooperativityand accounts for the sharp increase in Vo.Cooperativity means that allosteric enzymes display a threshold effect. Below

certain [S], there is little to no enzyme activity. After threshold is reached, enzyme

activity increases rapidly.

Signal Sensing capabilities:Positive effector binds to the R at a regulatory site and

stabilizes this form (R to S more likely). Negative effector binds to T and stabilizes it

(making R S binding less likely). Positive effector lowers the threshold

concentration of substrate needed for activity. Negative effector raises the

threshold.

Heterotropic effectors(regulatory molecules affect allosteric enzymes): activators:shift sigmoidal cure to the left. Inhibitors shift it to the right. ATP is an activator. CTP

is an inhibitor.

Homotropic effects(substrates affect allosteric enzymes): account for the

sigmoidal nature of the kinetics curve.

Sequential model:binding of substrate at one site influences the substrate binding

to neighboring sites without necessarily inducing a transition encompassing entire

enzyme. (negative cooperativity:binding of one substrate decreases the affinity of

other sites for the substrate)

Ensemble studies:experiments on enzymes.

Enzyme Mechanism and Inhibition Lecture 12 Chapter 8

Covalent catalysis:active site contains a reactive group, usually a powerful nucleophilethat becomes temporarily covalently modified in the course of catalysis. (proteolytic

enzyme chymotrypsin)

General Acid-Base Catalysis:a molecule other than water plays the role of proton

donor/acceptor. Chymotrypsin uses histidine residues as a base catalyst to enhance thenucleophilic power of serine.

Metal Ion Catalysis:metal ions can function catalytically in several ways. Either

serve as a electrophilic catalyst (stabilizing negative charge on reaction

intermediate). Metal ion may generate a nucleophile by increasing acidity of nearby

molecule. Or may bind to substrate, increasing the number of interactions with the

enzyme and thus the binding energy.


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Hemoglobin Lecture 13- Chapter 9

Hemoglobin is an allosteric protein. It is a component of red blood cells. Carries

oxygen from the lungs to the tissues and contributes to the transport of carbon

dioxide and hydrogen ions back to the lungs.

Both hemoglobin and myoglobin are oxygen binding proteins. Must be able to bind

and release oxygen

Myglobinis in the muscles, storage form of oxygen. Facilitates diffusion of the

oxygen to cellular sites that require oxygen and provide a reserve supply of oxygen

in times of need.

Hemoglobin behaves like allosteric. It displays cooperative behavior (sigmoidal

graph) Myoglobin behaves like M-M (hyperbolic curve).

Allosterichas to have more than one active site and be larger than primary

structure.

Myoglobin:single polypeptide chain consisting of mostly alpha-helices that are in a

globular structure. Globin fold. Can be oxy or deoxymyoglobin.

Myoglobin is a monomeric protein. Hemoglobin is tetrameric. Active site is on

the heme. Myoglobin only has 1 heme (oxygen binding site)

Heme:a prosthetic group that determines whether myo/hemoglobin is able to bind

to oxygen.

The heme group gives muscle and blood their distinctive red color. It consists of anorganic component and a central iron atom. The organic component, called

protoporphyrin, is made up of four pyrrole rings linked by methine bridges to form

a tetrapyrrole ring. Four methyl groups, two vinyl groups, and two propio-nate side

chains are attached. Under normal conditions, the iron is in the ferrous F2+

oxidation state. Binding sites at fifth and sixth coordination sites.

Hemoglobin: HbA (adult)

Heme: Fe-protoporphyrin IX has Fe and pyrrole rings surrounding it.

Heme plane is attached to proximal histidine (5thsite)(imidazole ring of a histidineresidue of the protein) for hemoglobin and myoglobin and distal histidine on

opposite side. In deoxyhemoglobin/deoxymyoglobin6thsite is unoccupied

(available for binding oxygen).

Hemoglobin in the lungs has saturation level of 98% and 32% in tissues.

Hemoglobin delivers more O2 to tissues than would myoglobin.


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Homotropic effect:disruption of the TR equilibrium by substrates. The

heterotrophic effect: disruption of the TR equilibrium by effectors

Allosteric Effector Molecules that bind Hb: H+,CO2, Cl-, metabolite, 2,3-

biphopshoglycerate (BPG)

All of these bind dexoxyHB better than oxy-Hb, thus promoting release of O2 fromHb

Fetal Hemoglobin:(HbF): must bind oxygen when the mothers hemoglobin is

releasing oxgen.

No sickle cell anemia on exam.

Hydrogen Ions and Carbon Dioxide promote the release of oxygenThe Bohr effect

More CO2, pH goes down

The regulation of xygen binding by hydrogen ions and carbon dioxide is called theBohr effectafter Christian Bohr, who described this phenomenon in 1904.

The oxygen affinity of hemoglobin decreases as pH decreases from the value of 7.4

found in the lungs, at 100 torr of oxygen partial pressure ( Figure 9.18), to pH 7.2

and an oxygen partial pressure of 20 torr found at active muscle.

The heterotropic regulationof hemoglobin by hydrogen ions and carbon dioxide

further increases the oxygen- transporting efficiency of this magnificent allosteric

protein.

Carbonic anhydraseis an enzyme abundant in red blood cells.

MCB EXAM 2 Lecture Notes

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