TBL 2 - Enzyme Kinetics Flashcards
What are cofactors?
cofactors are metals and small organic molecules (coenzymes)
What are tightly bound coenzymes called?
Prosthetic group
What are loosely bound coenzymes called?
Cosubstrates
What type of reaction is oxidoreductases?
Oxidation-Reduction reaction e.g. Lactate dehydrogenase ( LDH catalyses the conversion of lactate to pyruvate and back, as it converts NAD⁺ to NADH and back. )
What type of reaction is transferases?
Hydrolysis reactions (transfer of functional groups to water) e.g. nucleoside, monophosphate and kinase (NMP kinase)
What type of reaction is hydrolases?
Hydrolysis reactions (transfer of functional groups to water) e.g. chymotrypsin (digestive enzyme component of pancreatic juice acting in duodenum where it preforms proteolysis - breakdown of proteins and polypeptide)
- What type of reaction is lyases?
Additon or removal of groups to form double bonds. e.g. Fumarase
What type of reaction is isomerases?
Isomerisation (intramolecular group transfer) e.g. Triose phosphate isomerase ( Involved in a critical energy-producing process known as glycolysis. Interconverts dihydroxyacetone phosphate and D: -glyceraldehyde-3-phosphate.)
What type of reaction is ligases?
Ligation of 2 substrates at the expense of ATP hydrolysis e.g. Aminoacyl- tRNA synthetase ( an enzyme that attaches the appropriate amino acid onto its corresponding tRNA.)
What is FAS?
Fatty acid synthesis that catalyses synthesis of long chain saturated fatty acids.
What is B-ox?
B-oxidation pathway breaks down saturated fatty acids with an even number of carbon atoms.
What does enzyme catalyse the conversion of?
Substrate and Products
What is product formation determined as?
Function of time
When is product completely formed?
Formed until no net change in conc of substrate and product and reaction equilibrium is reached.
Enzymes catalyses reactions by (4 things)…
1) providing a reactive surface and a suitable environment.
2) Bringing reactants together and positioning them correctly so they easily attain their transitional state configurations. 3) Weakening bonds in the reactants.
4) Reducing the energy required for the transition state.
Enzyme vs inorganic catalyst…
Enzyme:
1) All chemically similar
2) Easily inactivated (liable)
3) Usually specific
4) Specially inhibited
inorganic catalyst:
1) Chemically different (metal, salts and acids)
2) stable to heat
3) often non-specific
4) Usually non-inhibited
Inhibition (3 things)…
- Inhibition
- UNCHANGED at end of reaction
- DO NOT alter final equilibrium
- DO alter RATE of reaction.
What does decreasing enzyme specificity depend on?
1) Absolute (urease)
2) Stereochemical prefers D over L
3) Group or function (aromatic, methyl group and phosphate)
4) Low
What is liable?
Easily disturbed - if severe leads to denaturing.
What is the optimum temp of enzymes (hint: body temp)?
37 C
- What is the optimum PH?
7
What is the pH of pepsin in the stomach?
2
What is a unit of enzyme activity?
A unit of enzyme activity is the amount of enzyme causing transformation of 1umol of substrate per min @ 25 C.
What is the definition specific activity?
The specific activity is the number of units of enzyme activity per mg of enzyme protein.
What is the definition of Katal (Kat)?
The katal (kat) is the amount of enzyme activity that transforms 1 mole of substrate per sec.
What does the enzyme reaction rate not alter?
Does not alter the final equilibrium position, but speed up attainment of equilbrium
What is (S)?
Substrate
- What is (E)?
Enzyme
What is the reaction rate?
Velocity
What happens when there’s an increase in enzyme conc?
Increasing enzyme conc will increase reaction velocity as long as there is enough substrate molecules to bind to.
What happens at Vmax?
At Vmax, available enzyme molecules are saturated with substrates. All active sites are occupied.
Turnover number of enzyme:
Vmax = K2[E]T
What does Vmax show?
Vmax shows the turnover number of enzyme if the concentration of active sites (E)T is known.
What is the turnover number of enzyme?
number of substrate molecules converted into product by an enzyme molecule in an unit time when the enzyme is fully saturated with substrate.
When S < Km (a,b,c,d)….
a) conc of free enzyme (E) almost equal to the total conc of enzymes (E)T
b) Velocity of catalysis depends on Kcat/Km , (S) and (E)T values.
c) under these conditions Kcat/Km is the rate constant for the interaction of S and E and can be used as a measure of catalytic efficiency.
d) Using Kcat/Km values an enzymes preference for different substrate can be determined.
What is the meaning of Vmax?
Enzyme can’t work any faster all active sites are occupied. Enzyme is occupied. - measured in conc per unit time.
What is the meaning of Km?
Km gives a measure of how much substrate is needed for significant catalysis to occur. - unit of measurement is MOLAR (M)
km is half of vmax
catalytic efficiently of an enzyme formula is ….
V0 = kcat/Km [S] [E]T
What is Micheails Menton equation?
u (velocity) = VMAX . [S] / Km + [S]
What is Km the measure of?
measure of affinity for the formation of the enzyme-substrate complex (ES)
What happens at a low Km?
lower the km then greater the affinity for formation of enzyme- substrate complex (ES)
How do we go from a hyperbolic curve to a straight line?
Inverting michealis-menton equation
lineweaver-burke plot
Lineweaver-burke plot allows for…
the Km and Vmax values to be accurately estimated
What in the Lineweaver-burke plot equation?
1/u = Km/Vmax . 1/[s] + 1/Vmax
What are Competitive inhibitors and what do they do?
Bind to active site through intermolecular bonds and so the binding is reversible allowing an equilibrium to occur between bound inhibitor and unbound inhibitor. If the substrate conc is increased it competes more effectively so inhibitor binding will be less effective
examples of competitive inhibitor…
Sulphonamides (antimicrobials), statins, diuretics used to control blood pressure and antidepressant.
What are Non-competitive inhibitors and what do they do?
bind to an allosteric binding site and inhibit the enzyme-catalysed reaction without affecting the strength of the substrate bonding.
- Inhibition would occur if the induced fit arising from the binding of the allosteric inhibitor distorts the active sit sufficiently to prevent the catalytic mechanism but has no effect on substrate binding.
Examples: non-competitive inhibitors of CYP2C9 enzyme include…
nifedipine, tranylcypromine, phenethyl, lithocyanate and 6-hydroxyflavone.
