Proteins and Enzymes Notes PDF

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Summary

BIOC0001 Proteins and EnzymesWhat is a Protein?Body = 16% protein21 amino acids made of carbon, oxygen, nitrogen, hydrogen and/or sulfur.Selenocysteine contains 1 sel atom.Key words: carboxyl, amino, alpha central carbon, side chain (only variable region, determines properties)Hydrophobic amino acid...

Description

BIOC0001 Proteins and Enzymes What is a Protein? Body = 16% protein 21 amino acids made of carbon, oxygen, nitrogen, hydrogen and/or sulfur. Selenocysteine contains 1 sel atom.

Key words: carboxyl, amino, alpha central carbon, side chain (only variable region, determines properties)

Hydrophobic amino acids have carbon rich side chains. You can also have hydrophilic and charged amino acids.

Primary structure = linear sequence of amino acids as encoded by DNA. Peptide bonds link the amino group nitrogen of one amino acid to the carboxyl group carbon of another. A water molecule is released to form the protein backbone. Secondary structure = -

Alpha-helix: right-handed coil stabilised by hydrogen bonds between amine and carboxyl groups of nearby amino acids. Beta sheets: hydrogen bonds stabilise two or more adjacent strands of amino acids.

Tertiary structure = 3D shape of protein chain determined by characteristics of amino acid in the chain (eg. Charged, hydrophobic/hydrophilic etc.). Globular proteins have a sheltered hydrophobic core. Membrane-bound proteins have hydrophobic amino acids clustered together on exteriors so that hydrophobic side chains can interact with the lipids in the membrane. Haemoglobin’s 3D shape forms a pocket to hold heme, a small molecule with an iron atom in the centre that binds to oxygen.

Quaternary structure = two or more polypeptide chains com together to form one functional molecule with several subunits. 4 subunits of haemoglobin cooperate so the complex can more easily pickup oxygen in lungs and release in body.

Space-filling diagram = shows all of the atoms that make up a protein Cartoon/ribbon diagram = shows organisation of the protein backbone and highlights alpha helices

Surface diagram = shows areas in protein accessible to water molecules

Most proteins are smaller than the wavelength of light (eg. Haemoglobin ~ 6.5nm)

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Defence – antibodies – flexible arms recognise and bind to pathogens; targeting for destruction. Communication – hormones – insulin. Small, stable, can easily maintain shape whilst travelling through blood, maintains blood glucose level. Enzymes – alpha amylase – starts digestion of starch in our saliva. Transport – Ca2+ pump, aided by magnesium, powered by ATP, moves Ca2+ back to sarcoplasmic reticulum after muscle contraction. Storage – ferritin – spherical protein, channels that allow Fe2+ to enter/exit depending on need. On the inside is a hollow space where Fe2+ can attach to inner wall and be stored as Fe3+ (a nontoxic form) Structure – collagen triple helix. Fibrils/fibres – skin and tendons.

CPK colouring based on plastic space filling models developed by Corey and Paulin and improved by Kultun: -

Hydrogen = white Carbon = black Nitrogen = dark blue Oxygen = red Sulfur = deep yellow Phosphorous = orange Halogens (F, Cl, Br, I) = light, medium, medium-dark and dark green

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Metals (Co, Fe, Ni, Cu) = silver

Amino Acids: Amino acids have a chiral centre – central carbon is bound to 4 different groups. This can produce L and D stereoisomers. Draw the Fisher projection of the amino acid – put the carboxyl group on top and side chain on bottom. If the NH3 is on the left, it’s L (eg. L-alanine):

Amino acids in proteins are usually L, but there are some exceptions (eg. Bacterial cell walls)

Selenocysteine (the active site of seleno proteins) and pyrrolysine (a methanogenic bacteria) are rarer than the 20 common amino acids. Encoded by stop codons. There are both 3 and 1 letter abbreviations for all 22 amino acids above – in a protein code, you would use the 1 letter abbreviation.

Amino acids which have nonpolar side chains (within proteins, stabilise structure, NOTE this is not the same as being uncharged, you can be non-polar and still charged): 1. Glycine, Gly, G – the side chain is just a H, so there is no chiral carbon (or enantiomer form). Found in loops (spaces inaccessible to other amino acids because they’re too big). 2. Alanine, Ala, A – CH3 side chain. 3. Valine, Val, V -

4. Isoleucine, Ile, I –

5. Leucine, Leu, L (with an example of naming carbons) –

6. Methionine, Met, M – one of only two sulfur containing amino acids

Polar, uncharged amino acids (more soluble in water because they can form hydrogen bonds): 1. Serine, Ser, S – contains hydroxyl group

2. Threonine, Thr, T –

3. Asparagine, Asn, N –

4. Glutamine, Gln, Q –

Polar, charged amino acids (normally on the surface of proteins): Positively Charged: 1. Histidine, His, H (takes part in enzyme catalysed reactions) –

2. Lysine, Lys, K –

3. Arginine, Arg, R –

Negatively Charged:

1. Aspartic acid, Asp, D (note the second carboxyl group but with a negative charge) –

2. Glutamic acid, Glu, E –

Non-polcisar aromatic amino acids (hydrophobic interactions, but tyrosine and tryptophan slightly more polar due to NH and OH. Allows absorption of UV light by some proteins): 1. Phenylalanine, Phe, F –

2. Thyrosine, Tyr, Y –

3. Tryptophan, Trp, W –

‘Special’ Amino Acids: 1. Proline, Pro, P (entire structure incorporated into side chain. Rigid, reduces structural flexibility of the protein. 5 membered ring) –

2. Cysteine, Cys, C –

The SH above can form disulphide bridges. 2x cysteine use this mechanism to form cystine:

The disulphide bridge formed is strongly hydrophobic and structural because it can form links both within a polypeptide chain and between different chains.

Soluble proteins have a hydrophobic interior and charged exterior. Membrane proteins: -

Lipids with the polar head and non-polar tail. Non-polar tails interact with each other.

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Integral proteins have a hydrophobic interior and a hydrophilic exterior. The hydrophobic interior will interact with the phospholipid tails. Peripheral membrane proteins are only on the surface of the phospholipid bilayer – they’re hydrophilic and only interact with the polar heads.

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Amino acid side chains can undergo modification: Modification type: Phosphorylation N-Glycosylation D-Glycosylation Hydroxylation Carboxylation Methylation Disulphide bond formation

Chemistry: Phosphate group added to R group Sugar added to NH group H on hydroxyl (OH) group is removed and replaced with sugar Hydroxyl group added to R group Carboxyl group added to R group Methyl group added to R group Oxidation

Amino Acid Titration Curves: Diprotic acids can donate two protons Diprotic bases can accept two protons. Eg. For alanine:

Applicable amino acid residues: S, T, Y N S, T P, K E R, K C

Zwitterions can both donate and accept protons, so can act as acids or bases

pK1 = pH when half of the COOH in the sample has been deprotonated. Is equal to the pH of COOH. The 1 stands for the fact that it’s the first group that is being deprotonated. pI = overall midpoint, all of the carboxyl groups have been deprotonated. Isoelectric point. Ala is a zwitterion at this point. pK2 = as above but for amino group. The flatter regions around pK 1 and pK2 are due to buffering

Titration curve for aspartic acid (3 ionisable groups – ionisable side group):

Approximations of pKa values to remember: Group/acid: Carboxyl group Aspartic and glutamic acid Histidine Cysteine Amino group Lysine and tyrosine Arginine

pKa: 2 4 6 8.5 9.5 10.5 12.5

In proteins, the ammonium and carboxyl groups form peptide bonds so cease to exist, so the only things to inform the charge of the proteins are the sidechains.

The pI is the isoelectric point, the pH at which the net molecule charge is zero. Calculate: -

For amino acids with two ionisable groups; it’s the mean of the two pK a values. For amino acids with three ionisable groups; it’s the mean of the two pK a values immediately around the zwitterion/pI (could be pK1 and pK2 as seen for aspartic acid, but could also be pK 2 and pK3 as seen in lysine).

Aspartic acid, glutamic acid, lysine, arginine and histidine all have three ionisable groups.

Peptide Bonds: Covalent bonding forms dipeptides:

R groups are NEVER used (only alpha groups – carboxyl and amino) The amino acids incorporated into the dipeptide are called residues

The peptide bond was studied in detail by Pauling and Corey, who analysed geometry and the structure of the bond in crystal protein structures. They found that the peptide bond is planar and rigid, due to resonance structures:

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Resonance is a way of describing bonding in certain molecules of ions by the combination of several contributing structures into a resonance hybrid (overall structure) in valence (outer shell electron) bond theory. The amide structure (same as on the left) has two resonance contributors:

Because the bond between the carbonyl carbon and the nitrogen has a partial double bond character, rotation is restricted around the bond, causing the planar rigidity. Generally, peptide bonds have the same trans configuration – the two alpha carbons of the connected amino acids are on opposite sides of the peptide bond. Rarely though, it can be cis:

The cis configuration gives rise to steric hinderance – clashes between groups attached to the alpha carbons. About 6% of proline residues (the weird ringed one) are in this form. A torsion/dihedral angle is formed by three bonds in a molecule and is the angle created in the middle bond (between the two outer bonds), eg:

Bond: Phi,

ᶲ

Psi, ᴪ Omega, ω

Atoms involved:

C-N-Ca-C N-Ca-C-N Ca-C-N-Ca

Angle: Free Free 180

Ramachandran – used computer models to systematically vary

ᶲ and ᴪ torsion angles, to find

which combinations are possible. Results are presented in a Ramachandran plot. Impossibilities because of atoms coming closer than the sum of their Van der Waals radii have been identified. Glycine has its own Ramachandran plot – because it only has H as its side chain, it can adopt more angles, due to reduced Steric Hinderance. However, proline (ring structure) is more restricted, so there are less allowed regions on the Ramachandran plot. Oligopeptides, Polypeptides and Proteins: You can have tripeptides and tetra (4) peptides Oligopeptides = 90% of R is bound

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Decrease [L] to 0.01um and [L]=

1 K , [RL]= 100 d

1 [ R] , so >>>[E0], so [S]~[S0]

K1 ( [E0] - [ES] ) [S0] = ( K-1 + K2 ) [ES]

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Rearrange

[ES] =

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K 1 [ E0 ] [ S 0 ] K 1 [ S0 ] +K 1 + K 2

Simplify

[ E0 ] [ S0 ] [ES] =

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Use Km =

[ES] =

V0 =

V0 =

[ S 0 ]+(

K−1 +K 2 ) K1 K −1+ K 2 K1

[ E0] [ S0 ] [ S 0 ]+K m

Combine with V0 = K2[ES]

K 2 [ E0 ][ S0 ]

[ S 0 ]+ K m Vmax=K2[E0] when enzyme is saturated

V max [ S 0 ]

[ S0 ] +K m

At a very low substrate concentration, most of the enzyme is in free form (uncombined). In this case, our rate is proportional to the initial substrate concentration. However, if we add more substrate, we can push the equilibrium towards ES formation

So when [S0] is much smaller than K m, [S0] becomes negligible in the denominator and we are in a linear range However, when [S0] is sufficiently high that most of our enzyme is in ES form, adding more substrate will not increase reaction rate. At this point, K m will be negligible in comparison to [S0], so V0 will approximate Vmax.

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V0 =

Vmax is the initial reaction velocity when the substrate concentration is high compared to K m Km is the substrate concentration resulting in half-maximal velocity The initial reaction velocity at any single substrate concentration is proportional to [E0]. So if we double [E0], V0 will also be doubled

V max [ S 0 ]

[ S0 ] +K m

can be rearranged to

Km 1 1 + = V 0 V max [ S0 ] V max

in order to provide a linear plot

called a Lineweaver-Burk plot. This was very useful before the time of computers:

We don’t use these anymore, we use specialised software. Also, this is problematic … 1. All data at very large substrate concentrations will be clustered near the origin 2. Experimental errors in the first order region will be very spread and amplified from those near the origin, so could potentially significantly determine how the line is drawn

More than one substrate binding event will still follow the same hyperbolic curve pattern as Michaelis-Menten, and can fit into the equation: V0 =

K cat [ E0] [ S 0 ]

[S 0 ] + K m

, where Kcat[E0]=Vmax and Kcat is the turnover number (the general rate constant

which describes limiting rate of an enzyme-catalysed reaction at saturation) For example … 1. In the presence of a competitive inhibitor:

2. Two substrate binding events, separated by irreversible transitions:

Some enzymes, however, do not follow Michaelis-Menten and instead follow the yellow on the graph below (eg. ATCase) which require much more complex equations:

Km (units = M) is a property associated with binding of substrate to enzyme. It is not just a simple measure of substrate affinity. When K 2 is small compared to K-1, Km is a fair approximation of substrate binding Kd = dissociation constant of EC =

K −1 K1

The

[ S0 ] Km

ratio is typically between 0.01 and 1.0 under physiological conditions; if our initial

substrate concentration is much higher than Km, our enzyme would be saturated under all conditions, so changes to [S] would not be detected. So [S0] cannot be much higher than K m, but it also can’t be much lower, otherwise most of the enzyme would be in the free state (waste of resources).

Vmax (units: ms-1) and Kcat (units: s-1) are properties associated with the enzyme’s ability to turn over substrate Kcat is equivalent to the number of substrate molecules converted to product in a given unit of time on a single enzyme molecule when the enzyme is saturated with the substrate (so we’re NOT interested in E+S here) Specificity constant = how efficiently an enzyme converts a specific free substrate into product =

K cat Km

. The higher the specificity constant, the more the enzyme prefers that substrate. We can

use it to compare different substrates with an enzyme.

Diffusion controlled limit is 108 to 109 m-1s-1. This is the general rate of diffusion. For some enzymes, specificity constant approaches the diffusion-controlled limits (eg. Acetyl cholinesterase, fumarase and beta-lactamase all have a specificity constant of 108m-1s-1. Most enzymes have a specificity constant above 105m-1s-1.

Rate enchancement =

∆G K cat =e K uncat

⊨uncat

−△ G RT

⊨cat

Ranges from 105 to 1017 Allows us to see how much the enzyme is capable of decreasing the activation energy for the reaction

U ) ml – An important measure of enzyme purity. Check it Specific activity (u/mg) = mg ) Total protein ( ml Totalactivity (

after trying to purify an enzyme! U = nmol/min – one unit is the amount of enzyme that catalyses the reaction of 1nmol of substrate per minute under standard conditions

Inhibition:

Irreversible inhibition = slow dissociation of enzyme-inhibitor complex. Tightly bound to enzyme (often covalent). E.g. group specific suicide. Reversible inhibition = rapid dissociation of enzyme-inhibitor complex. E.g. competitive, uncompetitive, non-competitive, mixed.

Competitive inhibition:

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Inhibitor resembles the substrate and binds directly to the active site, forming an enzyme inhibitor complex. So the substrate and inhibitor are ‘competing’ for the same site. Increasing inhibitor concentration will increase EI formation, decreasing ES due to reduced enzyme availability. If we hugely increase [S], we will move the position of equilibrium back to free enzyme production from enzyme inhibitor complexes, to reduce [S] Km is increased due to hindering binding of substrate, but Vmax is not influenced as this can still be achieved through hugely increasing [S] The alpha has been added to the Michaelis-Menten equation because it will influence K m

Example of a competitive inhibitor = methotrexate. Widely used in cancer treatment as a competitive inhibitor to dihydrofolate reductase, an enzyme which provides key metabolites for nucleotide biosynthesis (so RNA/DNA synthesis is blocked and the cancer cells die). The natural substrate of dihydrofolate reductase is dihydrofolic acid, which is structurally very similar to methotrexate.

Uncompetitive inhibition:

Inhibitor blocks the turnover of substrate into product, but cannot work unless an ES complex has first formed, maybe because … The inhibitor can bind on a different site that’s only accessible after substrate binding, or the site may be formed by the substrate, or the inhibitor may bind to groups on both the enzyme and the substrate The inhibitor and the substrate are not competing with each other here, so it’s uncompetitive However, this means that even if we increase [S], we cannot overcome the effects of uncompetitive inhibition Very rare form of inhibition Non-competitive inhibition: more common

There’s a permanent site on the enzyme that the inhibitor can bind to, regardless of whether a substrate is present or not

The dissociation constants for the enzyme inhibitor complex and the enzyme substrate inhibitor complex are the same, therefore K m is unchanged. For example, nevirapine. It’s a non-nucleoside reverse transcriptase inhibitor, another example of which is etravirine. They bind in a pocket in the P66 subunit close to the active site. Reverse transcriptase is used by HIV to replicate by basically copying its viral RNA genome into DNA. It’s a heterodimer – each dimer is composed of two subunits: P66 and P51.

Mixed inhibitors: similar to non-competitive; the inhibitor can bind to both enzyme and enzymesubstrate complex, but there is a different affinity for the substrate and inhibitor, so K i is not equal to Ki ’ . Km changes – dissociation constants decide whether this is an increase or a decrease.

In order to study inhibition, we must measure the conversion of substrate to product as a function of time. Draw progress curves for various substrate concentrations in the absence and presence of an inhibitor at fixed [E0] and plot all the data onto one graph to determine type of inhibition, K m, Vmax, Km,app and Vmax,app. Use this to then determine inhibition constants (Ki).

Direct inhibition plots:

Lineweaver-Burk plots:

Irreversible inhibition: Group-specific:

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Bind to specific side chains of amino acids (eg. Iodoacetamide inhibits cysteine proteases because it interacts with the catalytic cysteine residue). Another example is diisopropylfluorophosphate (DIPF or DFP) react with serine proteases such as trypsin and chymotrypsin. This was evaluated by the British for chemical warfare use during WWII, but never used. They were used as pesticides but are now banned because they’re highly toxic for non-target organisms.

Suicide (mechanism-based):

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Bind to th...