09/11/08Biochemistry: Protein Function Protein Function Andy Howard Introductory Biochemistry, Fall...

09/11/08Biochemistry: Protein Function

Protein Function

Andy HowardIntroductory Biochemistry, Fall 2008

11 September 2008

09/11/08Biochemistry: Protein Function of 33

Topics for today Zymogens and

Post-translational modification

Allostery Specific protein

functions Structural proteins Enzymes Electron transport

Specific functions (continued) Storage & transport

Proteins Hormones &

receptors Nucleic-acid binding

proteins Other functions

Distributions


Zymogens and PTM Many proteins are

synthesized on the ribosome in an inactive form, viz. as a zymogen

The conversions that alter the ribosomally encoded protein into its active form is an instance of post-translational modification

Bacillus amylo-liquifaciensSubtilisin prosegment complexed with subtilisinPDB 1spb2.0Å29.2+8.6 kDa


Why PTM?

This happens for several reasons Active protein needs to bind cofactors, ions,

carbohydrates, and other species Active protein might be dangerous at the

ribosome, so it’s created in inactive form and activated elsewhere Proteases (proteins that hydrolyze peptide bonds)

are examples of this phenomenon … but there are others


iClicker question 1

Why are digestive proteases usually synthesized as inactive zymogens?

(a) Because they are produced in one organ and used elsewhere

(b) Because that allows the active form to be smaller than the ribosomally encoded form

(c) To allow for gene amplification and diversity (d) So that the protease doesn’t digest itself

prior to performing its intended digestive function

(e) None of the above


iClicker question 2Which amino acids can be readily

phosphorylated by kinases? (a) asp, phe, gly, leu (b) ser, thr, tyr, his (c) leu, ile, val, phe (d) arg, lys, gln, asn (e) none of the above.


iClicker question 3Why are kinase reactions ATP- (or GTP-)

dependent, whereas phosphorylase reactions are not?

(a) To ensure stereospecific addition of phosphate to the target

(b) To prevent wasteful hydrolysis of product (c) Adding phosphate is endergonic; taking

phosphate off is exergonic (d) None of the above.


Allostery Formal definition:

alterations in protein function that occur when the structure changes upon binding of small molecules

In practice: often the allosteric effector is the same species as the substrate: they’re homotropic effectors

… but not always: allostery becomes an effective way of characterizing third-party (heterotropic) activators and inhibitors


What allostery means Non-enzymatic proteins can be

allosteric:hemoglobin’s affinity for O2 is influenced by the binding of O2 to other subunits

In enzymes: non-Michaelis-Menten kinetics (often sigmoidal) when the allosteric activator is also the substrate

v0

[S]


R and T states Protein with multiple substrate binding sites is

in T (“tense”) state in absence of ligand or substrate

Binding of ligand or substrate moves enzyme into R (“relaxed”) state where its affinity for substrate at other sites is higher

Binding affinity or enzymatic velocity can then rise rapidly as function of [S]

Once all the protein is converted to R state, ordinary hyperbolic kinetics take over


Other effectors can influence RT transitions

Post-translational covalent modifiers often influence RT equilibrium Phosphorylation can stabilize either the

R or T state Binding of downstream products can

inhibit TR transition Binding of alternative metabolites can

stabilize R state


Why does that make sense?

Suppose reactions are: (E)A B C D

Binding D to enzyme E (the enzyme that converts A to B) will destabilize its R state, limiting conversion of A to B and (ultimately) reducing / stabilizing [D]: homeostasis!


Alternative pathways• Often one metabolite has two possible

fates: B C D A H I J

• If we have a lot of J around, it will bind to the enzyme that converts A to B and activate it; that will balance D with J!


How does this work structurally?

In general, binding of the allosteric effector causes a medium-sized (~2-5Å) shift in the conformation of the protein

This in turn alters its properties Affinity for the ligand Flexibility (R vs T) Other properties

We’ll revisit this when we do enzymology


Classes of proteins Remainder of this lecture:

small encyclopedia of theprotein functions

Be aware of the fact thatproteins 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


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

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

CollagenmodelPDB 1K6F


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




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)


IUBMB Major Enzyme ClassesEC # Class Reactions Sample Comments

1 oxidoreductases Oxidation-reduction

LDH NAD,FMN

2 transferases Transfer big group

AAT Includes kinases

3 hydrolases Transfer of H2O

Pyrophos hydrolase

Includes proteases

4 lyases Addition across =

Pyr decar-boxylase

synthases

5 isomerases Unimolec-ular rxns

Alanineracemase

Includesmutases

6 ligases Joining 2 substrates

Gln synthetase

Often need ATP


Electron-transport proteins

Involved in Oxidation-reductionreactions 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


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


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 myoglobin


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)


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.

Not all hormones are proteins some are organic, non-peptidic moieties Others: peptide oligomers, too small to be proteins But some hormones are in fact normal-sized proteins.

Human insulinPDB 1t1k3.3+2.3 kDa


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


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!


Nucleic-acid binding proteins

Many enzymes interact with RNA or DNA But there are non-catalytic proteins that

also bind nucleic acids 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”

Human hDim1PDB 1pqnNMR struct.14kDa


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


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


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


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


What percentages do what? See fig. 5.32 in G&G 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


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

09/11/08Biochemistry: Protein Function Protein Function Andy Howard Introductory Biochemistry, Fall...

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Transcript of 09/11/08Biochemistry: Protein Function Protein Function Andy Howard Introductory Biochemistry, Fall...