BM210 - BLOCK B (not L1)

how do catalysts increase affect reaction? what is the transition state?

increases rate of reaction by lowerig activation energy (transition state)

• is not consumed in the reaction

• does not effect equilibrium

transition state is the unstable point that reactants must pass through to become products. → catalysts stabilises this by lowering the activation energy it occurs at

how is the active site formed?

Folding of the protein brings side-chains of various amino acids far apart in primary squence into close juxtaposition, forming an active site.

what is the process of an enzyme catlysed reaction?

1. substrates enter active site (Enz + S)

2. substrates are held by ionic interactions and hydrogen bonds → Enz-S complex

3. enzymes breaks/forms bonds in substrates converting to products → Enz-P complex

4. products are released (Enz + P)

5. ezymes remained unchaged and reaction can start again

reversible reaction

Enz + S     ⇌        Enz-S       ⇌       Enz-P        ⇌      Enz + P

how does the active site lower activation energy/stabilise transition state?

1. positions substrates in correct orientation/alignment → avoiding collison

2. complementary to transition state (not substrate necessarily) → stabilising the transition state

3. amino acid side chains of active site stabilise electron distribution through hydrogen bonding, ionic interaction, covalent interaction → stabilising transition state

4. substrate is strained (distorted) → stretching/bending pushed closer to transition state faster

what are the Non-covalent interactions between the substrate and the amino acid side-chains of the active site?

Øacidic groups (Asp, Glu) → ionic bonds

Ø basic groups (Lys, His, Arg) → ionic bonds

Ø hydrophilic interactions with –OH or (Ser, Thr, Tyr)

Ø hydrophilic interactions with –SH (thiol) or (Cys)

Ø hydrophilic interactions with amide groups (Asn, Gln)

Ø aromatic interactions (Phe, Tyr, Trp)

Ø hydrophobic interactions (Ala, Leu, Ile, Val, Met)

how do reactive groups at the active site catalyse?

Ødonating (break bonds) or withdrawing electrons (form bonds)- from amino acids

Ø stabilising or generating free radical intermediates - weak interactions and temporary moving of electrons

Ø forming temporary covalent bonds → (a transition state intermediate) - acyl-enzyme → lowers activation energy

example of a enzyme-substrate interation?

yeast henokinase from hexokinase (enzyme) + glucose (substrate)

what is the induced fit model?

the enzyme changes shape around the substrate - the enzyme is flexible not rigid

what are co factors?the 3 types and their involvement with enzymes?

co-factors are non-proteins molecules

1. metal ions (inorganic) - Mg²⁺, Zn²⁺, Fe²⁺, Cu²⁺ → stabilise negative charges

2. prosthetic groups (organic) - attached to enzyme covalent bonds → heme, lipoic acid

3. co-enzymes (organic) - binds loosely to enzymes - NAD+/H, FAD, coenzyme A → carry chemical groups or electrons

what is the difference between a holoenzyme and apoenzyme?

holo - enzyme + cofactors → catalytically active

apo - enzyme with no cofactors → cataclytically inactive

what are the 6 major classes of enzymes and action?

1. Oxidoreductases - redox - dehydrogenase

2. Transferases: transfer a chemical group from one substrate to another - kinase

3) Hydrolases: hydrolysis (water splits the bond) of C-O, C-N, O-P and C-S   bonds - phosphatases

4) Lyases: addition across a carbon-carbon double bond

 

5) Isomerases: intramolecular rearrangements

6) Synthetases: formation of bonds between two substrates

  frequently linked to utilisation of ATP

unit of enzyme activity

(EU) = 1 μmol min^-1

specific activity

acitivty per mg of protein - gives protein purity (μmol min^-1mg^-1)

hyperbolic reaction rate

• rate of product formed slows - - from denaturation, substrate depletion

isoelectric point (pI)?

is the pH at which the molecule has no net electrical charge.

michaelis constant

Concentration of substrate to achieve half the maximum

rate of the reaction is K_m

• Low Km → high affinity
  (only a small amount of substrate is needed to reach half Vmax)

• High Km → low affinity
  (requires more substrate to reach half Vmax)

Michaelis-Menten equation

ternary complex? (sequential mechanism vs ping pong)

enzyme holds both substrates simultaneously

sequential - Both substrates must be present at the same time at different active sites - increasing B increases afinity for A (substrates are dependent) → converging lines on Lineweaver–Burk plot:

ping pong (double displacement) - substrates bind one at a time - substrate becomes modified following binding of 1st substrate ( substrates are independent) → parallel lines on Lineweaver–Burk plot:

Lineweaver–Burk plot:

double reciprocal plot used to determine Km and Vmax - straigns hyperbolic mentalis menten curve to be more accurate

• Y-intercept = 1 / Vmax → vmax increases moving up

• X-intercept = –1 / Km → km increases moving right

allosteric enzymes

produces sigmoidal curve - cooperative binding

multiple subunits with identical active sites

eg cyclin dependent protein kinase

example of covalent modification of an enzyme?

phosphorylation of ERK2 at threonine and tyrosine results in a structural change of the activation loop exposing hydrophobic region

reversible vs irreversible inhibitors?

Øreversible inhibitors - non-covalent binding to ezyme, unspecific → blocks substrate binding

Øirreversible inhibitors (inactivators) - bind to enzyme covalently, are substrate analogues, part of reaction → transition state covalent intermediate does not break down

competitive inhibotr? (reversible?)

 competes with the substrate for binding at the active site -

REVERSIBLE - as they bind non covalently and siplaced with high conc of substrates

• km increases because substrate ability to bind decreases

mixed inhibitors

bind allosteric site of E or ES complex chanigng enzyme shape → loweing vmax and altering km

• can be competitive binding straight to E → km increases vmax decreases

• can be UNcompetitive binding ES → km decreases VMAX DECREASES

can be NONcompetititive - binds E and ES - unchanged km / vmax decreases

allosteric activator/ inhibitors ?

sigmoidal - dont follow michael mentalis

not km → k0.5 = substrate conc at ½ vmax

positive modulator/activator → lower k0.5

no modulator - k0.5 unchanged

negative modulator/activator → increased k0.5 - more substrate need to reach ½ vmax

aldehyde vs ketone

aldehyde - HC=O - end of a chain

ketone - C=O - in the middle of a chainhem

what makes ribose deoxyribose

missing OH on carbon 2

hemiacetyl formation ? in glucose - pyronose?

aldehyde group on C1 reacts with alcohol OH group on C5 joining to form a/b anomers ring form

a = OH below

b = OH above

anomeric carbon is c1

hemiketal?

hemiacetyl formation but a ketone group not aldehyde

seen in fructose - ketose

anomeric carbon is c2

Sorbitol

Formed by the reduction of the aldehyde group of glucose to a hydroxyl group.

→ CHO → CH2OH

sorbitol is a sugar alcohol - tastes very sweet

o vs n glysosylic bond - in dna?

O bond formed when anomeric carbon reacts with alcohol eg methanol or serine

→ C1 with H above and OCH3 below

N bond formed when anomeric carbon binds nitrogenous base or lysine

→ C1 with H above and NR2 below

in dna - phosbate bind with O-glycosylic bond on C5 and N-glycosylic bond on C3

are phosphorylated sugar negative? examples

yes → cant pass cell membrane without transporters

G6P - first step of glycolysis

DHAP - metabolism

GAP - oxidised in glycolis to make ATP

which sugar type is nutritionally important\?

hexoses - glucose , fructose, galactose

disaccharide formation

O-glycosilic bond between two monosacharrides

intrinsic sugars vs extrinsic

intrinsic - good sugars contained within plant cell walls

extrinsic - bad, free in solution eg plaque - provide food for bacteria

— lactose from milk is good though

3 most common disacharrides and their bond type plus isotype of maltose

sucrose (cane or beet sugar - made from one glucose and
               one fructose) - a 1-2 link

Trehalose – especially in mushrooms - a 1-1 link

maltose (made from two glucoses) - produced by germinating cereals eg barley via amalase - a 1-4 link

→ isotype is isomaltose — linkages at a1-6

what moleculaes make up starch? glycogen similarity?

Amylose – chain of glucose molecules (a-1,4)

Amylopectin – chain of glucose molecules (a-1,4), every 30^th glucose → branch to other glucose residues (a-1,6 — ISOMALTOSE!)

glycogen - similar to starch, but branch every 10^th glucose via a-1-6

non - starch polysachharides?

e.g. Cellulose (glucose linked b-1,4

lactose intolerance

lactaste hydrolyes lactose at b1-4 link into glucose and galactose

→ decreases in lactase activity with age means lactose is converted to lactate, methane and hydrogen gas → farts (methan and hydrogen) and diarrhoea (osmosis from lactate)

glycogen metabolism

storage form of glucose in liver mostly (feeds brain) and skeletal muscle → insulin = store glucose, glucagon = release glucose

reducing / non-reducing ends of polysaccharides

reducing - free anomeric carbon can donate electrons

non reducing - anomeric carbon involved in glycosyllic bond so cant reduce

what is more stable open chain or hemiacetyl?

hemiacetyl

acetyl def

molecule with two single bonded oxygens attached to the same carbon atom - ie c1 with O-glycosillic linkage seen in lactose (b1-4) → is the non reducing end

why doesnt glycogen have a reducing end?

final glucose residue is covalently bound to a protein termed glycogenin via tyrosin

what is glycogenin

glycosyltransferase dimer at core of glycogen

what enzymes does glycogen contain

glycogen synthesis (glycogenesis) and degradation (glycogenolysis).

→ act on non reducing ends n

post translational modifications of carbohydrates

glycolysation - helps glyco protein stability, folding, recognition, nutrient sensing

protein O-glycosylation at serine or threonine in the Golgi

protein N-glycosylation at asparagine (Asn-X-Thr/Ser motif) in the ER sugar added via n-acetylglucosamine (GLcNAc) THESE ARE N LINKED GLYCANS

proteoglycans

proteins with long glycosaminoglycan (GAG) chain - mostly sugar 95%

found in cartiladge and ecm for cushioning and lubrication

mucins

O glycosylated proteins 80% carbohydrates

create mucus

glucosamine?

amino NH2 group added to carbon 2 of glucose

n linked glycan structure and types

main structure - 2GLcNAc and 3 mannose petasacharride core

high mannose type - many additional mannoses

complex type - extra GLcNAc, galactose, sialic acid and fucose

what are glycerophospholipids

glycerol + 2 fatty acids + phosphate + head group - amphiphatic

†he main componens of membranes

what are the functions of each glycerophospholipid

sphingomylein and cholesterol lipids function

fatty acids

long hydrophobic hydrocarbon chain attatched to carboxiyl acid (COOH-) hydrophilic head

saturated - single bonds , tight packing and solid at room temp

unsaturated (mono,poly) - cisdouble bonds , loose packing → INCREASED MEMBRANE FLUIDITY

shingolipid structure

sphingosine basis - long chain amino alcohol → unsaturated hydrocarbon chain with alcohol and amino group to attavh another fatty acid

sphingosine + fatty acid = ceramide → building block for sphingolipids

sphingomylien structure and use

ceramide + phosphocoline head = sphingomylien

hydrophic tail (sphingosine + fatty acid chain at amino site)

phosphocholine polar head

• major membrane lipid in mylin sheath

cholesterol structure and function

a sterol → 4 fused hydrocarbon rings with OH head and hydrocabon tail

• amphiphatic - OH interacts with membrane surface and tail inserts into lipid bilayer

• regulates membrane fluidity - at high temp → stabilizes membrane (less fluid), at low temp → prevents packing (more fluid)

bacteriorhodopsin

transmembrane protein of a-helices that span hydrophobic region of membrane

how does phosphatidylinositol (PI) signal

phosphorylated by kinase at various points

how are glycerphosphopipids synthesised, what can phosphoditate synthesis?

in ER

1. glycerol 3 phosphate (formed from DHAP or glycerol in liver) binds activated (saturated) fatty acid (R1-CO-CoA) at carbon 1 → lysophosphatidate

2. second (unsaturated) fatty acid (R₂-CO-CoA) is added to carbon 2 → phosphaditate (phosphatidic acid) PA

FATES OF PA

(DAG) a second messenger → phosphatidic acid phosphatetase (PAP) hydrolyses PA into DAG (diacylglycerol) + phosphate group (removes phosphate)

then

ON ER → triacylglycerol synthesis from DAG → add fatty acyl COA via Diacylglcerol acyl-transferase = TAG stored in fat/ liver

OR → glycerophospholipid syntheiss from PA → CTP activates PA = CDP-DAG → phosphatidylinositol/glycerol (PI/PG)

OR CTP activates head group (choline/ethanolamine) → transferring to DAG = PC/PE phosphatidylcholine/ethanolamine

Respiratory distress syndrome

lack of PC on lungs fucks surfuctant (surface tension) of fluid that keeps aveoli open aloowing gas exchange → low PC means aveoli collapse meaning fucked breathing / blue fingers

how are sphingolipids modified by sugars

ceramides → form cerebrosides by adding polar sugar head group eg glucose to UDP (uridine triphosphate)

→ gangliosides adding another sugar

Gangliosides function

-important cell surface molecules Highly prevalent in nervous tissue

tay sachs

Inherited disorder which affects motor function, then vision, fatal by 3 yr

→ unable to degrade gangliosides in lysosomes

how does ceramide form sphingosine then spingosine 1 phosphate

ceramidease adds fatty acid to ceremide amino group forming sphinogsine

sphingosine kinase adds phospahte group → spingosine 1 phosphate

cholesterol biosynthesis step 1

1. acetoacetyl-CoA + acetyl- CoA → formation of HMG-CoA

2. HMG-CoA → mevalonate catalysed by HMG-CoA reductase in RATE LIMITING STEP

reductase converts 2NADPH → 2NADP+ and release CoA in process

step 2 cholesterol biosynthesis

malanovate is phosphorylated 3 times → decarboxylated forming Isopentenyl pyrophosphate (IPP)

→ IPP condenses to form squalene C5 → C10 → C15 → C30 (IPP→GPP→FPP→squalene)

→ squalene cyclises into lanosterol which is processed to cholesterol via removal of 3 methyl groups and double bond shifting

where does cholesterol synthesis?

LIVER/intestines

4 ways HMG-CoA is contolled?

1. rate of mRNA synthesis → when cholesterol is low SREBP (Sterol Regulatory Element–Binding Protein) (transcription facotr) enter nucleus and increases HMG-CoA production and vice versa when cholesterol is high

2. rate of translation → Translation of HMG-CoA reductase mRNA is inhibited by high levels of mevalonate and dietary cholesterol.

3. regulation by protein degredation → High cholesterol causes the HMG-CoA to bind to Insig proteins in the ER. This marks the enzyme for ubiquitin-mediated proteasomal degradation.

4. regulation by phosphorylation → Phosphorylation of HMG-CoA by AMPK stops cholesterol synthesis → dephosphorylation by glucagon activates HMG-CoA

what 2 forms of cholesterol are there

free cholesterol or in an esterified form in which it is linked to long-chain fatty acids

how is cholesterol transported in the body

lipoprotein particles - hydrophobic lipid core, surrounded by polar lipids and proteins

HDL (high density lipoprotein) = ‘good’ cholesterol

LDL (low density lipoprotein) = ‘bad’ cholesterol

high LDL?

increased risk of plaque formation in vessels and atherosclerosis.

bile salts - derivitive of cholesterol

Detergents (solubilise dietary lipids)

Synthesised in the liver - glycocholate, taurocholate

Stored in the gall bladder and released into small intestine

derivitive of cholesterol - steroidsd

Progestagens - fertilisation

Glucocorticoids Mineralocorticoids

Androgens – testosterone, progesterone

Oestogens

Derivatives of cholesterol – Vitamin D

vitimin D (cholecalciferol) → additon of 2 alcohol groups makes transcription facotr - calcitriol

statins

HMG-CoA inhibitors → aim to reduce cholesterol

what does amalase, lipase, and trpsin absorb

anerobic glycolysis - how is NAD regenerated

glucose is phosphorylated twice → fructose 1,6-bisphosphate

fructose cleaved into → dihydroxyacetone phosphate and glyceraldehyde 3 phosphate

glyceraldehyeide 3 phosphate → oxidised by GAPDH where 2NAD→2NADH then phosphoryliated twice where 2ADP→ 4ATP forming 2pyruvate PER GLUCOSE

LAck of NAD stops the oxidation step so regenerates by pyruvate +NADH → Lactate and NAD via lactate dehydrogenase

sites of control in glycolysis

-Hexokinase - glucose → glucose 6 phosphate (inhibited by its high conc glucose 6-phosphate)

- Phosphofructokinase - fructose 6 → fructose 1-6 (committed step; inhibition by: high ATP, low pH, citrate;
 activated by: AMP and fructose 2,6-bisphosphate)

- Pyruvate kinase PEP → pyruvate (ATP and alanine inhibit; fructose 1,6-bisphosphate activates)

phosphofructokinase 2 - regulated by?

bifunctional enzyme responsible for the synthesis and hydrolysis of
fructose 2,6-bisphosphate

has a kinase and phosphatase region - regulated by serine 460 by
protein kinase A

lactate - cori cycle

erithylocites lack mitochondria → no oxygen → pyruvate converted to lactate which muscles cant use → oxygen debt that needs cleared in liver with 6ATP (lactate→pyruvate→ glucose)

pyruvate oxidative decarboxylation

pyruvate → acetyl - COA (for tca cycle) by PDC (pyruvate dehydrogenase complex) in mitochondria

3 carbons → 2 carbons + SCoA + CO2

NAD → NADH

glycogen synthesis

IN LIVER AND MUSLCE

INITIATION - glycogenin (a glycosyl transferase) acts as primer

→ Glycogenin binds glucose from UDP-glucose to a hydroxyl group of tyrosine 194 via a-1-4 linkages

ELONGATION - glycoge synthase (GS) is phosphorylated by protein kinase A
  and glycogen synthase kinase 3 (GSK3) converting from a active form to b inactive form

(however, b is still active when a high level of the allosteric activator glucose 6-phosphate is present)

→ GS add branches to an existing chain of at least four glucose residues via a-1-6 linkages

UDP gluocse formation? - whay makes this irreversible ?

glucose-1-phosphate + UTP → UDP-glucose + 2 P_i

Spontaneous hydrolysis of the ~P bond in pyrophosphate (PPi (P-P))

importanc of branching ?

increased solubility of free ends in water

increases  terminal residues - the sites of action of glycogen
  phosphorylase and glycogen synthase

so increases synthesis and degredation of glycogen

fatty acid synthesis

decarboxylation reaction (releases CO2) in cytoplasm via acetyl CoA using ATP and NADPH adding 2 carbons at a time

acetyl CoA moves form mitochondria to cytoplasim by converting to citrate → acetyl coA

acetyl CoA +HCO3 → malonyl-CoA via acetyl coa carboxylase (ACC) using ATP → malonyl provides 2 carbon building blocks - irreversible

Transfer to acyl carrier protein (ACP) builds chain → +2C → REDUCE via NADPH → DEHYDRATW → REDUCE VIA NADPH resulting in Palmitate (16C)

what are fatty acids sotred as

as triacylglycerides (TG).

glycerol 3 phosphate + 3 fatty acids

Activation of fatty acids by CoA

Before TG synthesis, each fatty acid needs to be activated:

Thiol group of CoA → high energy thioester bond with COOH group of the fatty acid. - via Acyl CoA synthetase

Reaction driven by ATP  (2 high energy bonds used).

Making TG requires high energy investment.

what vitimins need fat , what polyunsaturated fatty acids need fat

A D E K

linoleic acid [C18:2] and linolenic acid [C18:3].

why do lipids (fatty acids ) need lypoproteins to be transported

they are hydrophobic and insoluble in aqueous
environments. 

fatty acid oxidation (b-oxidation)

1. activation - thiol of coenzyme A high energy thioester bond
   with the carboxylic acid group of the fatty acid. - via Acyl CoA synthetase using ATP and hydrolysing pyrophosphate

2. transport - Carnitine is an acyl-carrier that transports fatty acids into mitochondria

2. double bond created

3. water added to double bond

4. B oh → ketone group

5. ketone group attacked by coA splitting into fatty acyl coA and acetyl CoA

degredation of unsaturated fatty acids

double bond breaking requires - cis-D3-Enoyl CoA isomerase PLUS

2,4-Dienoyl CoA reductase

odd chain fatty acids

propionyl CoA → succinyl CoA via bicarbonate

gluconeogenesis - making glucose

Conversion of pyruvate into glucose (mainly in liver).

Major noncarbohydrate precursors are:

lactate, propionate, amino acids, glycerol

Where do they come from ?

- lactate, rate of glycolysis exceeds the rate of
  oxidative metabolism

- amino acids, breakdown of proteins

- propionate and glycerol, hydrolysis of triacylglycerols

glucose 6 phosphateinto glucose

glucose 6 phate → gluose via Glucose-6-phosphatase removing the phosphate group in liver and kidneys in the ER

fructose 1-6 biphosphate

fructose 1-6 phosphate — tetramer with mg /zn /mn as cofactors

AMP - low energy signal turns off converison

Fructose 2-6 phosphate - strong inhibitor activating glycolysis instead

citrate - activator

pyruvate converison

mitochondira 1. Pyruvate carboxylase activated by acetyl coA converts pyruvate → oxaloacetate (OAA) using ATP (adds CO2)

cytosol 2. Phosphoenolpyruvate Carboxykinase (PEPCK) - converts OOA → PEP + CO2 by adding phosphate using GTP

OAA in mitochondria can be reduce to malate or transaminated to aspartate to move into cytosol

transamination

transfer of amino group of amino acid to an a-ketoacid

Decarboxylations often drive reactions
that are otherwise highly endothermic !!!