Term 2 Lecture 3: Interacting Binding Sites, Catalysis (1) & Enzyme Classes Flashcards
Sigmoid binding curve and increased affinity for ligand binding
As seen for haemoglobin, indicates that binding sites on each subunit must interact with eachother so that as binding affinity increases more ligand is bound (i.e in Hb more O2 is bound)
Increase in affinity for ligand binding at each step is called positive cooperativity e.g. n>1 (neg coop also occurs but rarely)
Because binding sites on Hb are not directly in contact this is an example of allostery
Explaining sigmoidal curves AV Hill & GS Aldair
Hill equation:
Y= [L]^(n)/ Kd+[L]^n
Y= saturation 0-1
L= ligand conc.
Kd= dissociation constant
n= Hill coefficient
Hill equation is similar to 1 ligand equation. The Hill coefficient (n) defines cooperativity as a statistical dependence of one binding site on state of the other sites to give min. no. of binding sites.
E.g. The sigmoid binding curve for Hb can be analysed by Hill equation where n indicates “cooperativity” of binding sites and min no. Of binding sites that interact in Hb n= 4 but experiments never reach this value usually n~3 and is determined from the mid slope.
On a graph
X axis : log PO2
Y axis: log [YO2/(1-YO2)]*
*= nlog[O2] -log Kd
Hill and Adair did not cover structure change of ligand/protein on binding two models proposed in 1965 cover this
Model 1) Monod-Wyman-Changeux (MWC) model based on “all or nothing” switch in sub unit confirmation from T (tense) state to R (relaxed) state - really T is just diff from R
E.f. When no O2 is available T state strongly favoured almost all Hb tetramers are in T state
At low O2 conc. more tetramers are in R state(T tetramers also bind O2 at a lower rate)
High O2 conc. Hb approaches saturation almost all tetramers in R state
Binding O2 causes Haem ring structure to flatten shifting coordinates side chain of His F8 and pushing Val FG5 to one side. This has a knock on effect on confirmation throughout all 4 subunits. Changing from T to R state.
Changes in confirmation around the haem groups are transmitted from one subunit to neighbouring subunits through interfaces between them causing changes that affect all binding sites. Binding sites on other subunits now have flattened haem rings with greater O2 affinity - hence the sigmoid binding curve.
Model 2) Koshland-Nemethy-Filmer - KNF model. Subunits can shift sequentially - does not work so well for Hb but valid for other proteins
Interacting binding sites + evolution
Molecular evolution of globin oxygen binding proteins : the appearance of tetrameric haemoglobin is a feature of vertebrate evolution. The improved functional properties of this protein in loading O2 in lungs and discarding it in tissues - is the result of the sigmoidal curve caused by interactions between the subunits- necessary to support increased demands for O2 transport through blood vessel network (closed circulatory system) rather than via body cavity (open circulatory system) as in invertebrates.
Protein structure
Determines function - structure evolved to meet functional need.
Catalysis - what is a catalyst?
A catalyst is a substance that increases the rate (ie velocity) of a chemical reaction without itself being changed in the overall process. Enzymes are biological catalysts, almost all enzymes are proteins (ribozymes are RNA-based catalysts, ribosome is a ribozyme at its catalytic core)
Besides catalysis enzymes have 2 other properties vital for function:
Specificity: only act as catalysts for reactions involving specific molecules substrate(s) to products
Regulation: their catalytic activity can be altered often by molecules different from substrates
Heat (molecular motion) and thermal background heat
Heat= molecular motion
Thermal background heat
= ~RT= 8.314JK-¹mol-¹ x 298k
=~2.5 KJmol-¹
To work efficiently processes must have changes of >~4RT otherwise they can go backwards just as well as forwards (ie they are near equilibrium)
Motion can be of the whole molecule:
-translational- motion (diffusion in 1,2&3 dimensions)
- rotation
Or parts of the molecule can move relative to others:
-stretching and flexing (vibrations)
-rotation of part of the molecule
- both involve cov/non-cov interactions
Just out of “sight” (usually) are the quantum effects that define energy levels associated with molecular motifs
Uncatalysed Vs catalysed reaction
Uncatalysed:
reactants <-> collision complex <-> transition state <-> collision complex 2 <-> products
Catalysed
1) free enzyme E + A
2) E-A complex + B
3) E-A-B complex
4) transition state E+
5) E-C-D complex ( C is released)
6) E-D complex (D is released)
Return to (1)
Rate of chemical reaction is determined by
A parameter called the rate constant (K)
For reaction A+B-> products
Rate = K x (A)^alpha x (B)^beta
Where alpha and beta refer to order of reaction with reactants A & B respectively e.g. alpha = 1m
Rate constant (K) of a chemical reaction
Is determined by the activation energy Exact (units kJmol-¹) required to reach “transitional state”
Eact is constant for a given reaction under defined conditions.
Eact is related to rate by Arrhenius equation:
ln(k)=-Eact/RT +A
OR
Where A=collision frequency factor & T= temp. in K R= gas constant=8.314JK-¹mol-¹
In order for reaction to occur energy equivalent to Eact must be put into the system.
Normally activation energy comes from surroundings by collisions but reactions can also be driven by light energy (electromagnetic radiation) or by electrical energy or other energy sources.
How does a catalyst affect a reaction
Effects are brought about by the catalyst participating in the reaction in such a way as to decrease Eact (activation energy)
Decrease in Eact increases rate of reaction. There’s an exponential relationship between Eact and rate so a 10 fold decrease in Eact increases rate by a factor of >20,000!
K= Ae(-Eact/RT)
Note: reaction equilibrium ∆G is not changed by a catalyst
How enzyme catalysts work
Enzymes decrease Eact for a chemical reaction by binding the substrate(s.) Once bound the reaction may follow the same route as the uncatalysed reaction or the chemical mechanism may be different
Enzymes are catalysts that increase rates of specific reactions by factors of up to 10¹⁷ or more
E.g. CO2+H20<->H2CO3 (carbonic acid)
Catalysed by carbonic anhydrase, one of the fastest enzyme catalysed reactions known.
Allows functioning cells to adjust their metabolism on timescale required to respond to changes in surroundings. Like most enzyme catalysed reactions it can run forward or backward in Vivo depending on conditions (reversible reaction) determined by free energy change for the reaction (∆G) being small.
Note: some enzymes e.g. OMP decarboxylase produce extreme rate enhancements for reactions that hardly occur at all without catalysts at physiological temp.
Enzyme classes
1) oxidoreductases:
A(red) + B(ox) <-> A (ox) + B (red)
Oxidation - reduction
e.g. lactate dehydrogenase
Dehydrogenases, oxidases, peroxidases, reductases, monooxygenases, dioxygenases
2) transferases
A-B + C <-> A+B-C
Group transfer e.g. nucleoside, monophosphate, kinase (NMP Kinase)
3) Hydrolases
A-B+ H2O <-> A-H + BOH
Hydrolysis reactions e.g. chymotrypsin
4) Lyases (“synthases”)
A+B <-> AB
Addition/removal of groups to form double bonds e.g. fumarase
C-C Lyases, C-O Lyases, C-N Lyases, C-S Lyases
5) isomerases
A<-> Iso-A
Isomerisation - intramolecular group
Transfer e.g. triose phosphate isomerase
Epimerases, cos trans isomerases, intramolecular transferases
6) ligases
A+B+ATP <->A-B + ADP
Ligation of 2 substrates by ATP hydrolysis e.g. Aminoacyl -+ RNA synthetase
C-C ligases, C-O ligases, C-N ligases, C-S ligases
To carry out catalysis many enzymes need additional functionality
“conventional” enzymes are proteins made up of the “standard” set of 20 aa. Minimal range of side chain functionality seriously limits catalytic possibilities. To extend chemical functionality many enzymes contain additional atoms or compounds needed for catalytic activity.
Cofactor= atom or compound bound to an enzyme required for activity e.g. Mg²+ ion
Cofactors aid enzyme catalysis and include:
Coenzyme - organic compound used as a cofactor in enzyme (usually tightly bound e.g. in NADH)
Prosthetic group: non-removable coenzyme (usually cov bound e.g. haem)
Enzyme terminology
Apoenzyme: enzyme lacking cofactor (inactive e.g. opsin - binds to visual pigment retinal)
Holoenzyme: enzyme with bound cofactor(s) (active e.g. rhodopsin - opsin with retinol bound)
Metal ions often act as cofactors
and compounds produced by cellular metabolism often act as coenzymes
