protein functions; enzyme properties

09/15/2009Biochem: Protein Functions I

Protein Functions;Enzyme Properties

Andy HowardIntroductory Biochemistry,

Fall 2009 15 September 2009

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Biochem: Protein Functions I p. 2 of 40

Proteins and enzymes

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

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Plans for Today

Visualizing structure

The Protein Data Bank

Tertiary & quaternary structure

Protein Functions Structure-function relationships

Protein Functions PTM Allostery Classes of proteins and their roles

Enzyme properties Classes of enzymes

Enzyme kinetics

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How do we visualize protein structures?

It’s often as important to decide what to omit as it is to decide what to include

Any segment larger than about 10Å needs to be simplified if you want to understand it

What you omit depends on what you want to emphasize

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Styles of protein depiction All atoms All non-H atoms Main-chain (backbone) only One dot per residue (typically at C)

Ribbon diagrams: Helical ribbon for helix Flat ribbon for strand Thin string for coil

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How do we show 3-D? Stereo pairs

Rely on the way the brain processes left- and right-eye images

If we allow our eyes to go slightly wall-eyed or crossed, the image appears three-dimensional

Dynamics: rotation of flat image Perspective (hooray, Renaissance)

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Straightforward example Sso7d bound to DNA

Gao et al (1998) NSB 5: 782

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A little more complex: Aligning Cytochrome C5with Cytochrome C550

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Stereo pair: Release factor 2/3Klaholz et al, Nature (2004) 427:862

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Ribbon diagrams Mostly helical:

E.coli RecG - DNA PDB 1gm5

3.24Å, 105 kDa

Mixed:hen egg-white lysozyme

PDB 2vb10.65Å, 14.2kDa

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The Protein Data Bank

http://www.rcsb.org/ This is an electronic repository for three-dimensional structural information of polypeptides and polynucleotides

55660 structures as of 1pm today Most are determined by X-ray crystallography

Smaller number are high-field NMR structures

A few calculated structures, most of which are either close relatives of experimental structures or else they’re small, all-alpha-helical proteins

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What you can do with the PDB Display structures Look up specific coordinates Run clever software that compares and synthesizes the knowledge contained there

Use it as a source for determining additional structures

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Generalizations about Tertiary Structure Most globular proteins contain substantial quantities of secondary structure

The non-secondary segments are usually short; few knots or twists

Most proteins fold into low-energy structures—either the lowest or at least in a significant local minimum of energy

Generally the solvent-accessible surface area of a correctly folded protein is small

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Hydrophobic in, -philic out Aqueous proteins arrange themselves so that polar groups are solvent-accessible and apolar groups are not

The energetics of protein folding are strongly driven by this hydrophobic in, hydrophobic out effect

Exceptions are membrane proteins

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Domains Proteins (including single-polypeptide proteins) often contain roughly self-contained domains

Domains often separated by linkers

Linkers sometimes flexible or extended or both

Cf. fig. 6.36 in G&G

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Generalizations about quaternary structure Considerable symmetry in many quaternary structure patterns(see G&G section 6.5)

Weak polar and solvent-exclusion forces add up to provide driving force for association

Many quaternary structures are necessary to function:often the monomer can’t do it on its own

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Protein Function: Generalities

Proteins do a lot of different things. Why?

Well, they’re coded for by the ribosomal factories

… But that just backs us up to the question of why the ribosomal mechanism codes for proteins and not something else!

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Proteins are chemically nimble The chemistry of proteins is flexible

Protein side chains can participate in many interesting reactions

Even main-chain atoms can play roles in certain circumstances.

Wide range of hydrophobicity available (from highly water-hating to highly water-loving) within and around proteins gives them versatility that a more unambiguously hydrophilic species (like RNA) or a distinctly hydrophobic species (like a triglyceride) would not be able to acquire.

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Structure-function relationships

Proteins with known function: structure can tell is how it does its job Example: yeast alcohol dehydrogenase:Catalyzesethanol + NAD+ acetaldehyde + NADH + H+

We can say something general about the protein and the reaction it catalyzes without knowing anything about its structure

But a structural understanding should help us elucidate its catalytic mechanism

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Why this example?

Structures of ADH from several eukaryotic and prokaryotic organisms already known

Yeast ADH is clearly important and heavily studied, but until 2006: no structure!

We got crystals 8 years ago, but so far I haven’t been able to determine the structure



Yeast ADHPDB 2hcy2.44Å152 kDa tetramerdimer shown

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What we know about this enzyme

Cell contains an enzyme that interconverts ethanol and acetaldehyde, using NAD as the oxidizing agent (or NADH as the reducing agent)

We can call it alcohol dehydrogenase or acetaldehyde reductase; in this instance the former name is more common, but that’s fairly arbitrary (contrast with DHFR)

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Size and composition Tetramer of identical polypeptides Total molecular mass = 152 kDa We can do arithmetic: the individual polypeptides have a molecular mass of 38 kDa (347 aa).

Human is a bit bigger: 374 aa per subunit

Each subunit has an NAD-binding Rossmann fold over part of its structure

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Structure-functionrelationships II Protein with unknown function: structure might tell us what the function is!

Generally we accomplish this by recognizing structural similarity to another protein whose function is known

Sometimes we get lucky: we can figure it out by binding of a characteristic cofactor

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What proteins can do: I Proteins can act as catalysts, transporters, scaffolds, signals, or fuel in watery or greasy environments, and can move back and forth between hydrophilic and hydrophobic situations.

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What proteins can do: II Furthermore, proteins can

operate either in solution, where their locations are undefined within a cell, or anchored to a membrane. Membrane binding keeps them in place.

Function may occur within membrane or in an aqueous medium adjacent to the membrane

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What proteins can do: III

Proteins can readily bind organic, metallic, or organometallic ligands called cofactors. These extend the functionality of proteins well beyond the chemical nimbleness that polypeptides by themselves can accomplish

We’ll study these cofactors in detail in chapter 17

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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

PDB 3CNQSubtilisin prosegment complexed with subtilisin1.71Å; 35 kDa monomer

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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

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Protein Phosphorylation Most common form of PTM that affects just one amino acid at a time

Generally involves phosphorylating side chains of specific polar amino acids:mostly S,T,Y,H (and D, E)

Enzymes that phosphorylate proteins are protein kinases and are ATP or GTP dependent

Enzymes that remove phosphates are phosphatases and are ATP and GTP independent

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iClicker question 1Why 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

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iClicker question 2

Which 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.

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iClicker question 3Why are kinase reactions ATP- (or GTP-) dependent, whereas phosphatase 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.

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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

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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]

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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

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Glycogen phosphorylase BPDB 1XC798 kDa monomer1.83Å

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R and T state kinetics

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

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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

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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!

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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!

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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

protein functions; enzyme properties

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