Enzymes Flashcards
How do enzymes alter reactions?
Higher reaction rate under milder reaction conditions
Greater reaction specificity
Capacity for regulation
Lowers activation energy needed
What do enzymes not alter?
Don’t alter equilibrium point of reaction
Don’t alter overall free enery
What makes up an enzyme?
Protein molecules
And sometimes cofactors
What can the cofactors in enzymes be?
Inorganic molecules = metal ions
Coenzymes
Organic molecules = vitamins or derivatives
What are cofactors?
A non-protein chemical compound that is bound or unbound to the enzyme and is required for the enzymes catalytic activity
What happens to cofactors during reaction?
May be chemically changed by the reaction, so must by REGENERATED
What is a prosthetic group?
Bound cofactors to a protein
What’s a protein called with and without its prosthetic group?
Holoenzyme = active protein with prosthetic group
Apoenzyme = inactive protein because doesn’t have its prosthetic group
Describe what cytochrome and heme group relationship is
Heme is a prosthetic group of all cytochromes and is associated with Histidine residues
It is a prosthetic group because it is a tightly bound cofactors of cytochrome
Describe what G3PDH and NAD+ relationship is
NAD+ is the cofactor of glyceraldehyde 3-phosphate dehydrogenase
It is not bound to dehydrogenase permenantly = when reaction takes place is can then unbind and be used by another DH
What are the effects of pH on enzymes?
pH affects the ionization of polar groups on enzymes
What is the initial velocity and how is it measured?
Initial velocity is measured at the beginning of a reaction when the reverse reaction is insignificant
When starting out the products are 0 so probability of reversing the reaction is lower
What is the graph to calculate initial velocity?
Time = x axis
Product = y axis
What is the Michaelis-Menten equation?
Vo = Vmax [S] / [S] + Km
What are the axis for Michaelis-Menten plot?
x axis = [S]
y axis = Vo
How are Vmax and Km calculated from the Michaelia-Menten plot?
V max = max velocity, where it plateaus
Km = conc of substrate at half Vmax
When are initial velocity (Vo) and [S] proportional and independent of one another?
Vo is proportional to [S] when [S] is small = can see in graph there is a stright line
Vo is independent of [S] when [S] is large = graph starts curving
What are the axis for Lineweaver-Burk plot?
x axis = 1/[S]
y axis = 1/Vo
What are the important intersection on the Lieweaver-Burk plot?
y intercept = 1 / Vmax
x intercept = - 1 / Km
Gradient = Km / Vmax
What is the equation for 1/Vo?
(lineweaver-burk plot)
1/Vo = 1/Vmax + Km / Vmax [S]
What is the Km?
Concentration of a substrate at which the reaction velocity is half the maximal
Reflects the affinity of the enzyme for the substrate
What does having a higher Km mean?
The value of KM is inversely related to the affinity of the enzyme for its substrate.
High values of KM correspond to low enzyme affinity for substrate and vice versa
Define Vmax
Maximal rate of reaction (velocity)
Under saturation substrate conditions
What can we calculate from Vmax?
kcat (k_2)
What is kcat?
Catalytic constant = the number of maximum substrate molecules converted by the enzyme into a product per unit time when enzyme is fully saturated with substrate
ES»_space;> E + P
What is the catalytic constant also known as?
kcat is also known as the turnover number
What is the equation to calculate kcat?
Vmax = kcat x [ET = total enzyme concentration in our reaction]
What makes up total enzyme concentration in a reaction? [ET]
[ET] = [E] + [ES]
Describe the saturation conditions in terms of enzymes and substrates
All catalytic sites are saturated with substrate [ES] so no free enzyme [E] = 0
Thus, [Et] = [ES]
What does high kcat tell us?
The highest kcat shows the most catalytically efficient enzyme
kcat units = per second
What are the physiological conditions within the cell and what is calculated instead?
[S] is much lower than Km so kcat does not apply
kcat / Km is used to measure enzyme’s catalytic efficiency instead
Define diffusion-controlled limit
The frequency at which two molecules can collide
Enzymes which attain kcat/Km within the diffusion-controlled limit are considered to have attained kinetic perfection
What is the value of diffusion-controlled limit?
10^8 - 10^9 per M per s
What does Vo - [S] relationship look like for allosterically regulated enzymes?
Sigmoidal curve
What is the allosteric effect?
Binding of a ligand at one site affects the binding of another ligand at ANOTHER site of the same protein
Allosteric enzymes have two or more substrates
Define allosteric effectors
Ligands which bind to allosteric site and modulate the enzyme activity
Positive effectors = activation
Negative effectors = inhibition
What is a homotropic effector?
The substrate is itself
What subunit interactions usually occur in allosteric effects?
Interactions among subunits of OLIGOMERIC proteins (composed of more than one subunit)
What are the two forms of allosteric enzymes?
Taut = less active
Relaxed = more active
Name the two models of allosterism
Symmetry/Concerted model
Sequential induced-fit model
What is the symmetry/concerted model?
The ligand can bind to a subunit in either T or R conformation = molecular symmetry of the protein is conserved during the conformational change
Subunits must all exist at T or R conformation = no oligomers with both T and R state subunits
What is the sequential induced-fit model?
Ligand binding progressively induces conformational changes in the subunits, the greatest changes occur in those subunits that have bound ligands
Symmetry of oligomeric protein = not preserved
Cooperatively interactions = influence that binding of ligand to subunit has on neighbouring subunits
When is haemoglobin in T form?
No oxygen = Fe(II) is situated out of the plane of the heme because
1. doming of porphyrin skeleton
2. Fe-N (porphyrin) bonds are too long to allow Fe to lie in plane of porphyrin
When is haemoglobin in R form?
Interaction with oxygen
1. reduces doming
2. F-N bond shortens = Fe moves to centre of heme plane dragging along His and causing tertiary changes in polypeptide
As other subunits are closely coupled, structural change within one subunit will influence the structure of the other subunits
Give example of symmetry/concerted model enzyme
Aspartate transcarbamoylase (ATCase from E/coli) to make CTP
CTP is allosteric regualted of ATCase = assures that when there is a lot of product enzyme is inhibited so no more is made
