Chapter 10 continued PDF

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Summer 2019...

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Chemistry 474 – Biochemistry I Summer 2019

Chapter 10: Regulatory Strategies Chapter 12: Lipids and Cell Membranes

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Biochemistry Schedule • Online Quiz 5 – On Canvas today, due 11:59 pm Wed night • Team-Based Quiz 5 – During class Wed

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LECTURE OUTLINE

• proteolysis of zymogens – chymotrypsinogen and trypsinogen

• protease inhibitors – pancreatic trypsin inhibitor

• blood clotting cascade – intrinsic and extrinsic pathways

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Role of enteropeptidase • duodenal zymogens – must have coordinated activation – trypsin • common activator of all pancreatic zymogens – trypsinogen – chymotrypsinogen – proelastase – procarboxypeptidase – prolipase

– what activates trypsin? • enteropeptidase

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Inhibitors of proteases • pancreatic trypsin inhibitor – 6 kDa protein – binds tightly to trypsin active site in pancreas or pancreatic ducts • prevents acute pancreatitis • dissociation constant is 0.1 pM • standard free energy of binding is -18 kcal/mol • 8 M urea or 6 M guanidine hydrochloride do not dissociate this peptide-trypsin complex

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Inhibitors of proteases • pancreatic trypsin inhibitor is an effective substrate analog – inhibitor Lys-15 • interacts with Asp-189 in substrate specificity pocket to form salt bridge • many other additional H-bonds with trypsin main chain • Lys-15 carbonyl group in active site

– structure of inhibitor unchanged upon binding • Lys-15-Ala-16 peptide bond is l d t l t

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Other protease inhibitors • ฀1-antitrypsin – 53 kDa plasma protein – protects tissues from neutrophil elastase • blocks elastase more effectively than trypsin • binds nearly irreversibly to active site

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Other protease inhibitors

• ฀ 1-antitrypsin has a type Z mutation in humans

• genetic disorder produces ฀ 1-antitrypsin deficiency – Lys for Glu substitution at residue 53 – lowered liver secretion of ฀1-antitrypsin to 15% of normal in homozygotes – excess elastase destroys aveolar walls in lungs • digestion of elastic fibers and connective tissue proteins

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Blood clotting cascade • blood clot formation due to cascade of zymogen activations – two pathways • intrinsic pathway – endothelial lining rupture exposes anionic surfaces

• extrinsic pathway may be most crucial to blood clotting – trauma exposes tissue factor (TF) – integral membrane glycoprotein

– final common pathway • thrombin is protease that converts fibrinogen to fibrin 9

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Blood-clotting cascade Intrinsic pathway begins with activation of factor XII (Hageman factor) Extrinsic pathway triggered by trauma which releases tissue factor (TF) TF forms complex with factor VII to initiate cascade activating thrombin Inactive clotting factors = red Activated clotting factors = yellow

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Lecture Outline

•

cellular membrane components and properties

•

fatty acids –

common and systematic names

–

structures

•

3 common types of membrane lipids

•

phosphoglycerides based on glycerol

•

sphingomyelin based on sphingosine

•

glycolipids are also based on sphingosine

•

asymmetric distribution of phospholipids in membrane

•

cholesterol is a steroid

•

membrane lipids are amphipathic

•

membrane proteins

–

–

–

phospholipids, glycolipids, cholesterol

phosphatidylserine (PS), phosphatidylcholine (PC)

membrane formation, lipid vesicles, impermeable lipid bilayers

–

peripheral and integral

–

membrane spanning a-helices

–

channel proteins and b-strands

–

prostaglandin H2 synthase-1 (cyclooxygenase-1)

–

covalently attached hydrophobic groups anchor proteins

–

amino acid sequence predicts transmembrane helices

•

•

•

bacteriorhodopsin

bacterial porin

lipid and protein diffusions in membranes –

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lateral diffusion

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HIV particle exits an infected cell by membrane budding

Cellular membranes may spontaneously self-assemble

• •

•

Hydrophobic interactions Fatty acid tails of membrane lipids pack together Polar heads exposed on surface

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Features of biological membrane 1.

Sheet-like structures • closed boundaries – 2 molecules thick – Thickness ~ 60 Å (6 nm) – 100 Å (10 nm)

2.

Lipids and proteins • Lipid:protein mass ratio = 1:4 – 4:1

3.

hydrophobic and hydrophilic properties • form closed biomolecular sheets • lipid bilayers – barrier for polar molecules

4.

proteins with many functions – pumps, channels, receptors, enzymes

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ti

l t

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bli

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Fatty acid structures

• fatty acids - long hydrocarbon chains – varying lengths and degrees of unsaturation – terminate with carboxylic acid • C18:0 saturated fatty acid is octadecanoic acid – parent hydrocarbon is octadecane

• C18:1 with one double bond is octadecenoic acid • C18:2 with 2 double bonds is octadecadienoic acid • C18:3 is octadecatrienoic acid

• carbon numbering starts at carboxy terminus – carbons 2 and 3 are a and b, respectively – terminal methyl is w-carbon – cis-Δ9 • cis double bond between carbons 9 and 10

– trans-Δ2 • trans double bond between carbons 2 and 3

• C # may start at distal end – w-carbon methyl is number one

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• w-3 fatty acids

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Fatty acid structures • fatty acids in ionized carboxylate forms at pH 7 – palmitate (16:0) and not palmitic acid – oleate (18:1) and not oleic acid

• – •

–

– •

degrees of unsaturation fatty acids usually contain even number of carbons 14 to 24 carbons - biological range

16 and 18 carbon fatty acids most common

unsaturation has 1 or more double bonds separated by -CH2usually cis double bonds

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MEMBRANE LIPIDS The common types : 1.

Phospholipids

2.

Glycolipids

3.

Cholesterol 17

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Phospholipids • abundant in all biological membranes – composed of 4 components • one or more fatty acids • platform for fatty acid attachment • phosphate • alcohol attached to phosphate

– platforms • Glycerol • 3 carbon alcohol – Phosphoglycerides or phosphoglycerols » glycerol, 2 fatty acids, phosphorylated alcohol » C-1 and C-2 are esterified to fatty acid

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Alcohols and phosphoglycerides • major phosphoglyceride s – derived from phosphatidate

• major phosphoglycerides – phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), diphosphatidylglycerol (cardiolipin)

• ester bond between phosphate of

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Sphingomyelin and cerebroside • sphingomyelin – phospholipid derived from sphingosine • sphingosine is an amino alcohol with long unsaturated hydrocarbon chain • amino group of sphingosine backbone linked by amide bond to fatty acid • primary hydroxyl group of sphingosine esterified to phosphorylcholine

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Membrane lipids with carbohydrate • glycolipids – 2nd major class – sugar-containing lipids derived from sphingosine • sphingosine backbone amino group acylated by fatty acid • sphingosine primary hydroxyl linked to one or more sugars and not phosphorylcholine

– simplest glycolipid is cerebroside • single glucose or galactose residue

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Learning Check Which is correct? a) phosphatidylethanolamine contains choline b) sphingomyelin contains glycerol c) in sphingonyelin, the primary -OH group of sphingosine is esterified to phosphorylserine d) phosphatidate is the simplest phosphoglyceride e) phosphatidylcholine contains sphingosine

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Lipid based on steroid structure • cholesterol - third major type – steroid built from 4 linked hydrocarbon rings – hydrocarbon tail and hydroxyl group at opposite ends – orientation is parallel to phospholipids in membranes • hydr head

– absent animal

pholipid

rly all

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Ether lipids with branched chains • archaea membranes differ from eukaryotes or prokaryotes – – – –

non-polar chains in ether linkage to glycerol backbone glycerol stereochemistry inverted compared to phosphatidate alkyl chains are branched and saturated archaeal lipids resistant to hydrolysis (ether linkage) and oxidation (branched alkyl chains) – lipids help archaea withstand high temperature, high salt, low pH

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Phosphoglyceride model • membrane lipids – amphipathic molecules • hydrophilic and hydrophobic moieties

– phosphatidylcholine • roughly rectangular • hydrophobic fatty acid chains parallel • hydrophilic phosphorylcholine polar headgroup points in opposite direction to chains • sphingomyelin and archaeal lipids in similar conformations

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Phospholipids and glycolipids readily form bimolecular sheets • two limiting possibilities due to amphipathic nature – micelle • small structure < 200 Å (20 nm) in diameter

– lipid bilayer or bimolecular sheet • favored leaflet structure in aqueous media • extended dimensions of up to 107 Å (~1 mm) • 2 fatty acid chains in phospholipid or glycolipid are too bulky for micelle

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Spontaneous formation • phospholipids form lipid bilayers – self-assembly process that is rapid and spontaneous – hydrophobic interactions • major driving force for lipid bilayer formation • van der Waals forces between hydrocarbon tails – favor close packing of hydrocarbon chains

– hydrophilic interactions • polar head groups and water – participate in electrostatic and H-bonding interactions

• lipid bilayers are cooperative structures – many reinforcing, noncovalent interactions • predominantly hydrophobic interactions 27 • lipid bilayers are extensive compartmentalized self-sealing

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Phospholipids form lipid vesicles • lipid vesicles or liposomes – aqueous compartments enclosed by lipid bilayer – study membrane permeability or deliver drugs to cells – liposome formation • PC + aqueous medium + sonication di i i

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Molecular trapping • preparation of Glycontaining liposomes – PC + 0.1 M Gly + aqueous medium + sonication – 500 Å diameter liposomes with ~2,000 Gly – liposome separation from surrounding Gly medium • dialysis or gel-filtration chromatography

– Gly efflux gives liposome permeability

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Proteins and membrane processes • membrane proteins – chemical transport and information transfer across permeability barrier – protein content of membranes vary depending on function • myelin – Membrane (electrical insulator) is only 18% protein

• metabolically active membranes – plasma membranes are ~50% protein (pumps, channels, )

SDS-PAGE of membrane proteins A = erythrocyte plasma membrane B = retinal rod cell photoreceptor membrane C = muscle cell sarcoplasmic reticulum membrane 30

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Protein association with membrane • peripheral membrane proteins – electrostatic and Hbond binding to lipid head groups (c) – binding to integral membrane protein surfaces (d) – covalently attached fatty acid hydrophobic chain may anchor to membrane (e) – high ionic strength

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Mechanism of protein spanning • integral membrane proteins • common structural feature: membrane-spanning α helices • bacteriorhodopsin – archaeal membrane protein – uses light energy to transport H+ out of cell – H+ gradient drives ATP synthesis View from cytoplasmic side of – 7 perpendicular a-helices span membrane 45 Å width of cell membrane 32

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Primary sequence is important • bacteriorhodopsin – most membrane spanning a-helical residues are hydrophobic – nonpolar amino acids • in contact with hydrocarbon membrane core or with one another

Amino acid sequence of bacteriorhodopsin • Yellow regions are 7 a-helices • Red residues are charged 33

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