BMS02 - Molecules and Cells :evergreen_tree: (Enzymes and Coenzymes…
BMS02 - Molecules and Cells :evergreen_tree:
PH and Buffering
PH is a measure of H+ ion concentration. Acidity and alkalinity depend on 'free' protons and not those bound to anions.
Blood PH is maintained at a range of 7.3-7.4 and is highly regulated to prevent acidosis and alkalosis.
Most acids enter the circulation via breakdown of proteins, incomplete oxidation of fats, transport of Carbon Dioxide etc.
Majority of the body is made of water. The ionic product of water is [H+][OH-] = 1 x 10^-14 mol^2 dm-6. Water barely dissociates.
PH = -log[H+]
The Critical PH is the highest PH at which there is a net loss of tooth mineral approx. 5.5.
Lipid soluble molecules pass through membranes better when uncharged. For weak acids for example, this will be below PKA as there is a higher chance of association.
Buffers are systems which resist large PH changes either by releasing or binding to protons to maintain a stable PH. They consist of a mixture of a weak acid and it's conjugate base.
Buffers rely on PKA (acid dissociation constant) - PKA is the PH where a weak acid is half dissociated so there are equal amounts of acid to conjugate base.
The lower the PKA value, the more acidic a solution is and vice versa.
The Henderson-Hasselbach equation
is used to relate PH to PKA.
Buffers operate best at PKA
as if protons are added, the conjugate base may bind to it. If OH- is added, the weak acid can donate protons creating water leading to no major PH change.
Amino acids can act as buffers
but the zwitterion usually cannot unless it falls within the 'buffering region' but it more often than not occurs during an equivalence point. Almost all amino acids are poor physiological buffers due to their buffering ranges except
One of the most important physiological buffers in the body is
. It is effective as it has a large number of histidine residues; the buffering range of histidine is higher in Hb than free Histidine because neighbouring R-groups affect the PKA value.
Deoxyhaemoglobin is a better buffer than oxyhaemoglobin
due to the Haldane effect.
Amino Acid and Protein Structure
Proteins have very unique structures and this specificity affects their functions.
Proteins are encoded by genes to form linear polymers with a specific three dimensional structure.
Proteins are encoded by genes and produced by the translation of mRNA. They often have additional organic groups e.g. sugars, phosphates etc. to make them more suited to function.
Protein structure is hierarchical going from primary to secondary to tertiary and sometimes even to quarternary level of structure and arrangement.
Primary structure is the sequence of monomers building up to make the polymer. The monomers are residues (amino acids which are coded by codons which are degenerate) linked by peptide bonds. The assembly occurs in the ribosomes via dehydration reactions.
Tertiary structure is the further folding of the protein to make it more specific and is non-linear. Quaternary structure is the interaction of more than one polypeptide chain, stabilised in the same way as tertiary e.g. Haemoglobin.
Tertiary structure is mainly dependent on hydro-interactions with hydrophobic groups positioned on the inside and hydrophilic groups on the outside. Giving the protein a 3D structure. If hydrophilic residues are in the centre instead, this forms a pore/channel for the movement of hydrophilic molecules.
Tertiary structure is stabilised by hydro-interactions, hydrogen bonds and disulphide bonds. Disulphide bridges are caused by the interaction of cysteine molecules to form intra-chain links.
Secondary Structure is both linear and non-linear.
The peptide bond is fairly special. It has a partial double bond character which leads to very limited rotation around it.
The bond is stabilised by a 'resonance' with delocalised regions of electrons above and below the bond, making it planar in shape.
The peptide bond creates rigid portions with rotations of bonds allowed on either side. These rotations rely on phi/psi (dihedral) angles illustrated by the Ramachandran plot. Some prefer an alpha helix arrangement or a beta pleated sheet. Proline has the highest steric interference and causes the turns in an helice and dislikes the helix arrangement the most.
Some amino acids have limited rotations and are called 'steric groups' and so prefer certain arrangements to others. This is one of the main drives of secondary structure.
Secondary structure is stabilised by dihedral angles, hydrogen bonding and electrostatic interactions.
An alpha helix
(right-handed) has 3.6 residues per turn with each residues 0.15nm apart.
A beta pleated sheet
is stabilised by inter-molecular hydrogen bonds between several sheets.
Collagen is an example of a filamentous (fibrous) protein.
Often synthesised via it's pre-cursor pro-collagen. Has approx, 3 residues per turn with each 0.31nm apart.
Pro-collagen is a left-handed triple helix of glycine, proline and hydroxyproline. Mature collagen also may contain hydroxylysine. With the coil maintained by hydrogen bonding.
Collagen production is influenced by Vitamin C. Vitamin C acts as a co-factor for prolyl-4-hydroxylase which is responsible for hydroxylating proline. So having scurvy influences collagen production.
Amelogenin is an important protein in dentistry. It allows for proper tooth formation and the attachment of enamel giving teeth its unique hardness.
All amino acids have a chiral centre except glycine. The chiral carbon is surrounded by an R-group, a hydrogen, an alpha-carboxyl and an alpha-amino group.This asymmetry allows for optical isomerism (enantiomers) etc.
The R-group usually dictates the properties of the amino acid.
If the R group is
alkyl or aromatic
(no polar), it will be
. If the R group is
, acidic or basic, the residue will be
. Acidic and basic amino acids dictate the charge of a protein.
are Glycine, Alanine, Valine, Leucine, Isoleucine, Methionine and Proline. These groups have prominent van der Waals force which are hydrophobic interactions.
are Glutamate and Aspartate whilst R=Basic are Lysine, Histidine and Arginine.
are Phenylalanine and Tryptophan.
are Tyrosine, Serine, Threonine, Cysteine, Glutamine and Asparagine.
Enzymes and Coenzymes
Enzymes are biological catalysts which speed up reaction rates without altering the final equilibrium. They speed up reactions by reducing the activation energy by putting reactants in the correct orientation, in a reactive state and also stabilises the transition state.
Enzyme Catalysis is very specific. The active site has a defined 3D structure; the site is specific enough that it can distinguish between optical isomers. This specificity is important so pathways remain uninterrupted.
This specificity also relates to the reactions they catalyse which allows enzymes to be classified.
: transfer of functional groups from donors to acceptors.
: addition of water to cleave a bond (hydrolysis) e.g. chymotrypsin with peptide bonds
How Chymotrypsin works
: add O2 or remove 2H. Also involved in the transfer of electrons e.g. a dehydrogenase
: addition of groups to a double bond or the formation of a carbon double bond by group removal.
: transfer of functional groups within the same molecule.
: using ATP to catalyse the formation of new covalent bonds.
: transfer of an inorganic phosphate to an acceptor. Whilst a
deals with the transfer of a phosphate group from ATP to a acceptor.
Enzymes are proteins with unique 3D structure maintained by many weak bonds/interactions e.g. hydrogen bonding, electrostatic interactions and hydro-interactions.
Being reliant on these weak interactions for structure (particularly the active site which stabilises the transition state) makes enzymes very sensitive to environmental changes.
Temperature; most enzymes have an optimum temperature in which reaction velocity will peak but if it is greatly exceeded it can lead to denaturation.
PH; different enzymes work best at different PH values. PH usually interferes with hydrogen bonding.
has five isoenzyme forms as it is a tetramer. These forms can be distinguished using electrophoresis/chromatography and are used to detect myocardial infarction as the 'heart form' will be most abundant in the blood.
Isoenzymes are enzymes with different protein structures which catalyse the same reaction. They are coded by different genes and are often found in different cells or organs altogether e.g. Glucokinase and Hexokinase.
Some enzymes require co-factors or co-enzymes to function at maximum efficiency.
Some inorganic elements such as Fe2+ and Fe3+ act as co-factors usually by accepting or donating electrons e.g. Cytochrome Oxidase in the electron transport chain.
Co-enzymes usually accept/donate protons and electrons acting as 'energy carriers' such as NAD and FAD.
- how the rate of an enzyme-catalysed reaction varies dependent on the concentration of substrate, inhibitors and co-factors.
is usually measured as the change in product conc. divided by change in unit time.
Most enzymes follow saturation kinetics - this is shown on a Michaelis-Menten plot. The plot assumes the enzyme is saturated with substrate.
At lower substrate conc.; the curve is almost linear meaning the reaction rate is proportional to substrate conc. (1st order kinetics). At high substrate concentrations, reaction rate is independent of substrate concentration as the curve plateaus (zero order).
The plot assumes that substrate conc. greatly exceeds enzyme conc. The concentration of enzyme-substrate complex does not change so the rate of forward and backwards reactions are equal. Initial velocities are used.
The Michaelis-Menten model is based on this
In order to calculate initial velocity, we use the Michaelis-Menten equation.
Km is the substrate concentration of an enzyme-catalysed reaction where the initial velocity is half of the maximum
. If Vo = 0.5Vmax; Km = [S].
Different enzymes have different Km values. Km can in certain instances be used as a measure of substrate affinity only if; k2<<k-1. k2 is often rate-limiting so if it is very small Km will become Kd (the dissociation constant for ES). k2 is rarely smaller than k-1 so Km is not a good measure for affinity.
Kcat is the turnover number
. The number of substrate molecules converted into product molecules in a given unit time per enzyme molecule. The best way to compare efficiency is using the specificity constant: Kcat/Km.
The Lineweaver-Burke Plot is a rearrangement of the Michaelis-Menten equation to give a linear line. Allows us to find the values of Km and Vmax.
can be described
as competitive and non-competitive
Competitive inhibitors block an enzyme's active site.
Non-competitive inhibitors interfere with the catalytic mechanism somehow and can be reversible or irreversible.
With competitive inhibition, the value of Km changes but Vmax does not. On a Lineweaver-Burke plot, the line will be steeper as gradient = Km/Vmax.
Competitive inhibitor diagram
Non-competitive inhibition will not change the value of Km. The reaction will not reach Vmax regardless of increasing substrate concentration. The line will be steeper but the value of km will be the same.
Non-competitive inhibitor diagram
A popular example of inhibitors are ACE inhibitors; used in the prophylaxis of hypertension and heart failures.
is the addition or removal of a covalent bond to alter the enzyme. A good example is the removal and addition of a phosphate group to activate and inactivate an enzyme. This can be done by a kinase/phosphatase. Adenylylation (ADP to PPi) and Uridylylation (UDP to PPi) are other examples.
Induction or repression of enzyme synthesis
is the most long term means of regulation. This is usually controlled by hormones e.g. after feeding, insulin encourages the production of glucokinase and pyruvate kinase.
is when another molecule binds to the enzyme (not at the active site). This can either have a positive or negative effect on an enzyme. These allosterically affected enzymes show a sigmoid curve. An example is ATP and Citrate of Phosphofructokinase.
Electrical events happen on the either side of membranes and are measured in different ways using an electrode.
The Resting Membrane Potential
is in itself maintained by a electrochemical gradient.
The resting membrane potential (Vm) is approximately
. The potential is dictated by [K+] and [Na+]. There is a high conc. of K+ intracellularly whilst there is a high conc. of Na+ extracellularly.
The value is rather negative and very close to Ek (equilibrium potential of Potassium) as the membrane is 50 times more permeable to Potassium so it has a larger bearing.
The equilibrium potential is the membrane potential/voltage a cell needs to be at to prevent the movement of a particular ion down its concentration gradient so essentially for it to not move creating an 'equilibrium'. So at Ek, Potassium ions will not leave the cell down it's concentration gradient.
Ek = -90mv and ENa = +50/60mv
At constant Vm, there is no net change in the number of ions as leak of ions out is equal to the number of ions in.
If the membrane becomes permeable to a particular ion, the electrochemical gradient will drive Vm towards the equlibrium potential for that ion.
The Electrochemical gradient is formed by the balance of osmotic work to electrical work.
A standard osmotic concentration gradient is measured by [Concentration out]/ [Concentration in] for cations and the inverse for anions.
Electrochemical gradients are reliant on the ability of the membrane to act as a
is the ability of the membrane to store ionic charges on it's surface and this is the basis of electrical work.
As potassium is being driven out of the cell along its concentration gradient, anions (e.g. Cl-) are electrostatically attracted to it and create a '
' effect. Ions then separate and line up along eitherside of the membrane.
The basis of the osmotic work is the establishment of concentration gradients. The Sodium-Potassium pump uses the hydrolysis of ATP to pump three K+ ions into the cell and two Na+ ions out of the cell against their concentration gradients. Thus, setting up these steep concentration gradients with large concentrations of Potassium inside and little outside with the inverse happening for Sodium. This then creates a osmotic driving force.
osmotic work is equal to electrical work
we get the equilibrium potential. This is dependent on
membrance permeability and conductance for an ion
Equilibrium potential for an ion is calculated using the Nernst equation.
The Goldman-Hodgkin-Katz equation considers the relative permeability of ions and can be used to calculate Vm.
Driving force can inform you whether an ion is being driven along or against its concentration gradient gradient.
Driving force = Vm - Eeq
So e.g. for Potassium. -70mv -( -90mv) = +20mv. So this the charge driving potassium down it's concentration gradient out of the cell.
A positive answer is driving an ion out of the cell whilst a negative answer is driving an ion into the cell.