Protein Functions II; Intro to Carbohydrates

09/14/2010Biochem: Functions II; Carbohydrates I

Protein Functions II;

Intro to Carbohydrates

Andy HowardIntroductory Biochemistry,

Fall 2009 17 September 2009

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Biochem: Functions II; Carbohydrates I

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Proteins and carbohydrates

Proteins perform a variety of functions, including acting as enzymes.

Sugars are vital as energy sources, and they also serve as building blocks for lipid-carbohydrate and protein-carbohydrate complexes

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Plans for Today

Classes of proteins and their roles

Enzyme properties Classes of enzymes

Classification schemes

Sugar Concepts Review of chirality

Monosaccharides Oligosaccharides Glycosides

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Classes of proteins

Reminder:proteins can take onmore than one function A protein may evolve for one purpose

… then it gets co-opted for another

Moonlighting proteins (Jeffery et al, Tobeck)

Arginosuccinate lyase /Delta crystallinPDB 1auw, 2.5Å206kDa tetramer

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Structural proteins Perform mechanical or

scaffolding tasks Not involved in chemistry, unless you consider this to be a chemical reaction:(Person standing upright) (Person lying in a puddle on the floor)

Examples: collagen, fibroin, keratin

Often enzymes are recruited to perform structural roles

CollagenmodelPDB 1K6F

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Enzymes Enzymes are biological catalysts, i.e. their job is to reduce the activation energy barrier between substrates and products

Tend to be at least 12kDa (why?You need that much scaffolding)

Usually but not always aqueous Usually organized with hydrophilic residues facing outward

hen egg-white lysozymePDB 2vb10.65Å, 14.2kDa

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

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Many enzymes are oligomeric Both heterooligomers and homooligomers ADH: tetramer of identical subunits

RuBisCO: 8 identical large subunits, 8 identical small subunits



PDB 2hcy: tetramer

PDB 1ej7: 2.45Å8*(13.5+52.2kDa)

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IUBMB Major Enzyme ClassesEC # Class Reactions Sample Comments

1 Oxidoreductasese.g. 1.1.1.27

Oxidation-reduction

LDH NAD,FMN,metals

2 Transferasese.g. 2.6.1.1

Transfer big group

AAT Includes kinases

3 Hydrolasese.g. 3.6.1.1

Transfer of H2O

Pyrophos-phatase

Includes proteases

4 Lyasese.g. 4.1.1.1

Addition across =

Pyr decar-boxylase

synthases

5 Isomerasese.g. 5.1.1.1

Unimolec-ular rxns

Alanineracemase

Includesmutases

6 Ligasese.g. 6.3.1.2

Joining 2 substrates

Gln synthetase

Often need ATP

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Enzymes have 3 features Catalytic power (they lower

G‡) Specificity

They prefer one substrate over others

Side reactions are minimized Regulation

Can be sped up or slowed down by inhibitors and accelerators

Other control mechanisms exist

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EC System 4-component naming system,sort of like an internet address

Pancreatic elastase: Category 3: hydrolases

Subcategory 3.4: hydrolases acting on peptide bonds (peptidases)

Sub-subcategory 3.4.21: Serine endopeptidases

Sub-sub-subcategory 3.4.21.36: Pancreatic elastase

Porcine pancreatic elastasePDB 3EST1.65 Å26kDa monomer

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Category 1:Oxidoreductases General reaction:Aox + Bred Ared + Box

One reactant often a cofactor (see ch.7)

Cofactors may be organic (NAD or FAD)or metal ions complexed to proteins

Typical reaction:H-X-OH + NAD+ X=O + NADH + H+

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Category 2:Transferases

These catalyze transfers ofgroups like phosphate or amines.

Example: L-alanine + -ketoglutarate pyruvate + L-glutamate

Kinases are transferases:they transfer a phosphate from ATP to something else

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Category 3:hydrolases

Water is acceptor of transferred group

Ultrasimple: pyrophosphatase:Pyrophosphate + H2O 2 Phosphate

Proteases, lipases, many other sub-categories

HO-P-O-P-OH

O

OO-

O-

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Category 4:Lyases

Non-hydrolytic, nonoxidative elimination (or addition) reactions

Addition across a double bond or reverse

Example: pyruvate decarboxylase:pyruvate + H+ acetaldehyde + CO2

More typical lyases add across C=C

C=C

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Category 5: Isomerases

Unimolecular interconversions(glucose-6-P fructose-6-P)

Reactions usually almost exactly isoergic

Subcategories: Racemases: alter stereospecificity such that the product is the enantiomer of the substrate

Mutases: shift a single functional group from one carbon to another (phosphoglucomutase)

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Category 6: Ligases

Catalyze joining of 2 substrates,e.g.L-glutamate + ATP + NH4

+ L-glutamine + ADP + Pi

Require input of energy from XTP (X=A,G)

Usually called synthetases(not synthases, which are lyases, category 4)

Typically the hydrolyzed phosphate is not incorporated into the product

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iClicker quiz, question 1

Collagen is a structural protein. Collagenase catalyzes the hydrolysis of collagen under appropriate circumstances. It is:

(a) an enzyme (b) a hormone (c) a receptor (d) a nucleic-acid binding protein

(e) there’s no way to tell from the information provided.

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iClicker quiz, question 2 Suppose a membrane protein has 4 transmembrane helices and 2 aqueous domains, one at the N-terminal end and the other at the C-terminal end. Assuming the N-terminal aqueous domain is in the cytoplasm, where would the C-terminal aqueous domain be?(a) in the cytoplasm(b) in the membrane(c) in the extracellular space(d) no way to tell

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iClicker quiz, question 3

Which IUBMB enzyme category would collagenase fall into?

(a) ligases (6) (b) oxidoreductases (1) (c) hydrolases (3) (d) isomerases (5) (e) none of the above.

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iClicker quiz, question 4 Triosephosphate isomerase, whose structure we discussed last week, interconverts glyceraldehyde-3-phosphate and dihydroxyacetone phosphate. What would you expect the approximate G value for this reaction to be?

(a) -30 kJ mol-1 (d) 0 kJ mol-1

(b) 30 kJ mol-1 (e) no way to tell.

(c) -14 kJ mol-1.

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Electron-transport proteins Involved in Oxidation-reduction

reactions via Incorporated metal ions Small organic moieties (NAD, FAD)

Generally not enzymes because they’re ultimately altered by the reactions in which they participate

But they can be considered to participate in larger enzyme complexes than can restore them to their original state

Recombinant human cytochrome cPDB 1J3SNMR structure11.4kDa

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Sizes and characteristics

Some ET proteins: fairly small Cytochrome c Some flavodoxins

Others are multi-polypeptide complexes

Cofactors or metals may be closely associated (covalent in cytochromes) or more loosely bound

AnacystisflavodoxinPDB 1czn1.7Å18.6 kDa

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Storage and transport proteins Hemoglobin, myoglobin classic

examples “honorary enzymes”: share some characteristics with enzymes

Sizes vary widely Many transporters operate over much smaller size-scales than hemoglobin(µm vs. m): often involved in transport across membranes

We’ll discuss intracellular transport a lot!

Sperm-whale myoglobinPDB 1MTJ1.7Å 18kDa monomer

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Why do we have storage proteins? Many metabolites are toxic

in the wrong places or at the wrong times Oxygen is nasty Too much Ca2+ or Fe3+ can be hazardous

So storage proteins provide ways of encapsulating small molecules until they’re needed; then they’re released

T.maritimaferritinPDB 1z4a8*(18 kDa)

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Hormones Transported signaling molecules,secreted by one tissue and detectedby receptors in another tissue

Signal noted by the receptor will trigger some kind of response in the second tissue.

They’re involved in cell-cell or tissue-to-tissue communication.

Human insulinPDB 1t1k3.3+2.3 kDa

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Not all hormones are proteins

Some are organic, non-peptidic moieties

Others: peptide oligomers, too small to be proteins Oxytocin: CYIQNCPLG Angiotensin I: DRVYIHPFHL Some are cyclic (COO- terminus and NH3

+ termini hydrolytically ligated)

But some hormones are in fact normal-sized proteins.

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Receptors Many kinds, as distinguished by what they bind:

Some bind hormones, others metabolites, others non-hormonal proteins

Usually membrane-associated: a soluble piece sticking out Hydrophobic piece in the membrane sometimes another piece on the other side of the membrane

Membrane part often helical:usually odd # of spanning helices (7?)

Retinal from bacteriorhodopsinPDB 1r2nNMR structure27.4 kDa

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Why should it work this way? Two aqueous domains, one near N terminus and the other near the C terminus, are separated by an odd number of helices

This puts them on opposite sides of the membrane!

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Nucleic-acid binding proteins Many enzymes interact with RNA or DNA

But there are non-catalytic proteins that also bind nucleic acids

Human hDim1PDB 1pqnNMR struct.14kDa

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Non-catalytic nucleic acid binding proteins

Scaffolding for ribosomal activity Help form molecular machines for replication, transcription, RNA processing: These often involve interactions with specific bases, not just general feel-good interactions

Describe these as “recognition steps”

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Scaffolding(adapter) proteins A type of signaling protein(like hormones and receptors)

Specific modules of the protein recognize and bind other proteins:protein-protein interactions

They thereby function as scaffolds on which a set of other proteins can attach and work together



Human regulatory complex(Crk SH2 + Abl SH3)PDB 1JU5NMR structure

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Protective proteins Eukaryotic protective proteins: Immunoglobulins Blood-clotting proteins(activated by proteolytic cleavage)

Antifreeze proteins



E5 Fragment of bovine fibrinogenPDB 1JY2, 1.4Å2*(5.3+6.2+5.8) kDa

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Other protective and exploitive proteins

Plant, bacterial, and snake-venom toxins

Ricin, abrin (plant proteins that discourage predation by herbivores)



Synthetic Abrin-APDB 1ABR2.14Å29.3+27.6 kDa



Vibrio cholerae toxin A1 + ARF6PDB 2A5F2.1Å21.2+19.3 kDa

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Special functions

Monellin: sweet protein Resilin: ultra-elastic insect wing protein

Glue proteins (barnacles, mussels) Adhesive ability derived from DOPA crosslinks

Potential use in wound closure!



Dioscoreophyllum MonellinPDB 1KRL5.5+4.8 kDa

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What percentages do what? 42% of all human proteins have unknown function!

Enzymes are about 20% of proteins with known functions (incl. 3% kinases, 7.5% nucleic acid enzymes)

Structural proteins 4.2% Percentages here reflect diversity, not mass

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Protein Functions

Fig.15 from Venter et al. (2001), Science 291:1304

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Carbohydrates

These are polyhydroxylated aldehydes and ketones, many of which can exist in cyclic forms

General monomeric formula (CH2O)m, 3 m 9 With one exception (dihydroxyacetone) they contain chiral centers

Monomers and small oligomers: highly soluble Can be oligomerized and polymerized Oligomers may or may not be soluble Most abundant organic molecules on the planet

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How do we measure solubility for very soluble compounds? (Note: this is not a serious chemical topic: it’s an example of how statistics can be abused…)

The assertion is that, with highly soluble compounds like sugars, it’s difficult to use conventional approaches to compare their solubilities

The suggestion is that we might use the amount of time it takes to dissolve (for example) 50g of solute in 100mL of cold water: if it’s fast, the solute is more soluble than if it’s slow.

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Solubility measured by dissolution time

Assertion: more polar groups means shorter dissolution time for a given class of compounds

# of polar groups

Time required for dissolution

1

2

3

4

5

6

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What if we extrapolate to n=6?

We get a negative dissolution time!

That is, the solid goes into solution 6 seconds before we put it in the water!

This causes serious psychological problems (what if I change my mind?) and philosophical problems (is this pre-ordained?)

# of polar groups

Time required for dissolution

1

2

3

4

5

6

Observed points

Extrapolated point

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Whose idea is this? Isaac Asimov, that’s who!

“The endochronic properties of resublimated thiotimolene”:Astounding Science Fiction, March1948

My point: extrapolations and other misuses of statistics are dangerous

Benjamin Disraeli (popularized by Mark Twain):There are three kinds of untruth:lies, damn lies, and statistics.

Okay: let’s get back to the science.

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Aldoses & ketoses If the carbonyl moiety is at the end carbon (conventionally counted as 1), it’s an aldose

If carbonyl is one carbon away (counted as 2), it’s a ketose

If it’s two or more carbons from the end of the chain, it’s not a sugar

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Simplest monosaccharides Glyceraldehyde and dihydroxyacetone

Only glyceraldehyde is chiral:D-enantiomer is more plentiful in biosphere

All longer sugars can be regarded as being built up by adding-(CHOH)m-1 to either glyceraldehyde or dihydroxyacetone, just below C2

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How many aldoses are there? Every -(CHOH) in the interior offers one chiral center

An m-carbon aldose has (m-2) internal -(CHOH) groups

Therefore: 2m-2 aldoses of length m For m=3, that’s 21=2; for m=6, it’s 24=16.

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How many ketoses are there? Every -(CHOH) in the interior offers one chiral center

An m-carbon ketose has (m-3) internal-(CHOH) groups

Therefore: 2m-3 ketoses of length m

For m=3, that’s 20 = 1; for m=6, that’s 23=8.

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Review: stereochemical nomenclature Stereoisomers: compounds with identical

covalent bonding apart from chiral connectivity

Enantiomers: compounds for which the opposite chirality applies at all chiral centers

Epimers: compounds that differ in chirality at exactly one chiral center

One chiral center: enantiomers are epimers. > 1 chiral center: enantiomers are not epimers.

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Example: 2 chiral centers Chiral centers u,v; compounds A,B,C,D

Compound Stereo @ u

Stereo @ v

Enantio-morph of

Epimer of

A + + D B,C

B + - C A,D

C - + B A,D

D - - A B,C

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Properties

Enantiomers have identical physical properties (MP,BP, solubility, surface tension…) except when they interact with other chiral molecules

(Note!: water isn’t chiral!) Stereoisomers that aren’t enantiomers can have different properties; therefore, they’re often given different names

Protein Functions II; Intro to Carbohydrates

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