enzymes Flashcards
what are co-factors?
small molecules that are not part of the enzyme, but are required for activity
what is an enzyme lacking a co-factor called?
apoenzyme
what is the complex of enzyme and co-factor called?
holoenzyme
examples of co-factors?
inorganic co-factors: metal ions, zn2+, organic co-factors: vitamin c for collagen
do enzymes alter position of equilibrium?
no, they increase rates of forward and backward reactions
what is kcat?
the number of molecules of S that one active site can convert into product per second
how is complementarity between active site and S achieved?
high specificity, enzymes can differentiate between optical isomers, positional isomers and presence/ absence of functional groups
who proposed the ‘lock and key’ model?
emil fischer, 1890s
who proposed the ‘induced fit’ model?
daniel koshland, 1954
what did daniel koshland propose?
that S causes change in 3D relationship of active site and these changes bring catalytic groups into correct orientation for reaction
when was structural evidence for induced model produced?
1970s
how does a reaction of S to P proceed?
through a transition state, which is neither the substrate or the product
Properties of the transition state (X‡) in catalytic reactions?
requires a high Ea (ΔG‡) to achieve X‡, unstable high-energy combination of reactants, can either form products or fall apart to reactants
how do enzymes lower the ΔG‡ for X‡ to form?
non-covalent interactions (van der waals, H bonds, etc.) form between S and active site when ESC formed, releasing e and lowers ΔG‡, forming X‡.
how is Ea lowered for reactions with chymotrypsin?
aspartic acid, histidine, serine brought together during tertiary structure formation, lowering Ea
function of chymotrypsin?
protease, uses a wide range of proteins as S (non-specific)
what is covalent catalysis?
active site contains reactive group, which temporarily is attached to S
example of covalent catalysis?
reactive Ser on chymotrypsin attaches to substrate
what is acid-base catalysis?
molecule other than water acts as a proton acceptor or donor
example of acid-base catalysis?
chymotrypsin uses reactive His as base catalyst, which deprotonates + enhances reactive Ser
how does the catalytic amino acid triad of chymotrypsin activate the active site?
Asp H bonds with His, holding it in place, His H bonds with Ser, activating it
reaction mechanism steps of chymotrypsin?
S docks onto active site, induced fit, catalytic triad brought into close proximity to S, enzyme folds around S, target peptide bond is oriented close to catalytic Ser, attacks bond, acyl-enzyme X‡ resolved by Nu attack by H2O, releasing N-terminal of S, regenerating enzyme
what is enzyme kinetics?
study of enzyme-catalysed reaction rates
Equilibrium of enzyme reaction?
E + S ⇌ ES ⇌ E + P
What does V = k1[S][E] mean?
rate of reaction is proportional both to the concentration of substrate and enzyme
E + S -> (k1 above ar.) ES
rate of formation of ES depends on the [E] and [S], and is governed by the rate constant (k1)
E+S⇌ k1ES⇌k2 E+P
k-1 for ES to E + S
k-2 for E + P to ES
rate constants showing the equilibrium between forming and degrading ESC
When was Michaelis-Menten equation produced?
1913
What is the Michaelis- Menten equation?
Vo = (vmax*[S])/(KM+[S])
What is Vo?
the initial rate of reaction
What is Km?
[S] that gives 1/2 maximum reaction rate - half of active sites are occupies by S
How did Michaelis and Menten investigate enzyme kinetics?
1: Used a low [E], [S] > [E], showed that little change in [S], rate remained constant, [P] production linear when [S] > [E].
2: Determined initial rate (Vo) by finding gradient at early time points, passing through 0
Why did Michaelis and Menten use early time points to find Vo?
at early time points, [P] is low, so little P + E -> ES reverse, ignoring k-2
this simplifies overall reaction to:
E + S ⇌ (k1,k-1) ES -> (k2) E,
3 unknown constants left, simpler equation to work with
What were the findings of Michaelis and Menten?
After calculation Vo at varying [S], they found that Vmax is never reached due to hyperbolic relationship between [S] and Vo.
What was the ‘steady state’ argument?
there is a period where the formation of ESC = breakdown of ESC
Who and when proposed the steady state argument?
Haldane and Briggs, 1925
How to find Km from graph?
Guesstimate where Vmax is, find Vmax/2, which = Km
Physiological consequences of high and low Km?
mitochondrial aldehyde dehydrogenase has a low Km (high Vmax), efficient at low [acetylaldehyde] (small consumption of alcohol), can break down smallest amount of acetylaldehyde to acetate efficiently.
cytosolic ALDH requires high [acetylaldehyde] for Vmax, less efficient at lower alcohol consumption
What and when did Lineweaver and Burk find?
1934, rearranged M-M equation to y = mx + c, plotting 1/Vo over 1/[S], gives LINEAR transformation, where c = 1/Vmax.
What is the x axis intercept in a lineweaver-burk plot?
-1/Km
1/[S] = -1/Km
What is the y intercept in a lineweaver-burk plot?
1/Vmax
Drawback of using lineweaver-burk plot?
Measuring at low [S], gives large errors, altering line of best fit, so alters estimate of Km.
But, Vmax value the same
Why know value of Vmax?
helps us determine kcat,
kcat = Vmax / [E]
kcat constant under given conditions
How does increasing temperature alter enzyme activity?
Increase = more collisions = faster rate, however increasing past optimal = denaturing reversibly, but too high = irreversible denaturation
Increasing temperature equilibrium?
Eactive ⇌ Einactive -> Ex
(Ex permanently denatured)
Do all enzymes work at optimal temperature?
No, enzymes work suboptimally, if they worked at optimal t, too close to thermal denaturation (death)
How does altering pH change rates of reaction?
pH alteration alters ionisation status of groups on S and active site of E = alters efficiency of how active site finds S.
Can affect both Km and Kcat
What is Km?
[S] when half of enzyme active sites are occupied. gives half Vmax
Irreversible inhibitors: action of organomercurial compounds?
Organomercurial reacts with reactive Cys, -SH residue and permanently denatures enzyme.
Non-specific = very toxic
Irreversible inhibitor - nerve gas mechanism?
Diisopropylphosphofluoridate (DIPF) reacts with reactive Ser in active site, = permanently denatures acetyl cholinesterase = paralysis, death
Example of nerve gas?
Novichok, family of nerve agents inhibiting acetylcholinesterase, 2018 poisoning
How does addition of a competitive inhibitor affect rate?
Decreases initial rate, increasing the Km - more substrate to occupy 1/2 active sites to get to 1/2 Vmax.
Vmax stays the same (same y intercept)
How can competitive inhibitors bind to the enzyme’s active site?
They structurally mimic the structure of the substrate
Medical application example of competitive inhibitor?
Enalapril used as a competitive inhibitor, used to restrict vasoconstriction and lowers blood pressure, inhibiting Angiotensin-converting enzyme, which increases blood pressure
How does addition of a non-competitive inhibitor affect rate?
Reduces initial rate, does not compete for active site, so same Km as the amount of [S] needed does not change. Vmax is altered (different y intercepts)
Non-competitive inhibitor binding sites?
Binding at a site which isn’t active site = causes allosteric alteration (change in shape). This makes enzyme less efficient
What is a negative regulator of enzyme activity? Example?
an inhibitor effector molecule inhibiting enzyme activity by binding to an allosteric binding site and causing a conformational change, so less S can bind to active site
e.g. CTP production limiting ATCase activity
What is a positive regulator of enzyme activity? Example?
an activator effector molecule which increases enzyme activity by binding to an allosteric site, causing conformational change in enzyme, allowing substrate to bind more easily.
E.g. ATP promotes ATCase activity once [ATP] is high
How does having negative regulator CTP and positive regulator ATP help in reaction controlled by ATCase?
The regulators balance out the concentrations of purine and pyrimidines in a cell
What happens to ATCase when CTP binds to an allosteric site?
CTP binds the regulatory subunits and stimulates a conformational change in the enzyme so it’s in the T (tense) state - LOW ACTIVITY
What happens to ATCase when ATP binds to an allosteric site?
ATP binding the regulatory subunits causes conformational change of enzyme into R (relaxed) state - HIGH ACTIVITY
What does allosteric regulation display?
non-Michaelis-Menten kinetics: it shows cooperativity between multiple identical active site - fine regulation of activity over a narrow range of [S]. (sigmoidal kinetics of S binding)
What enzyme is allosterically regulated in glycolysis?
Pyruvate kinase (PK) regulated by allosteric interactions with FBP (glycolysis intermediate) which is positive effector (glycolysis promoted) and ATP which is negative effector (promoting gluconeogenesis).
Effect of allosteric regulation in metabolic reactions?
Can regulate the flux of metabolites through pathways (can change what reactions will occur, what will be produced)
Why is glyphosate used as a weed killer?
Inhibits EPSP synthesis, so essential amino acids cannot be made (Trp, Phe, Tyr)
What is Roundup ready?
genetically modified seeds which are resistant to the glyphosate herbicide, allowing for more effective crop growth
how were the roundup ready crops genetically modified?
bacterial gene resistant to glysophate was fused in frame to a chloroplast signal peptide into plant DNA.
significance of chloroplast signal peptide in genetically engineering crops?
directs the glysophate resistant gene + resultant protein into the chloroplast
how did the roundup ready crops survive the herbicide?
the glysophate resistant gene was translated into CP4 ESPS synthase, which gave resistance to glysophate, allowing for essential amino acids to continue being synthesised
Advantages of using roundup ready crops?
Reduce tilling to remove weeds, reduced soil erosion, increased profits for large farming industries
Disadvantages of roundup ready crops?
lower yield, expense of herbicide, increased herbicide resistance in weeds?
What does chymosin do in milk micelles + why important for cheese industry
Cleaves protein in the micelle, which clots the milk, allowing time for digestion (low pH needed). Clotting first part of making hard cheese
Why is recombinant chymosin (FPC) used in the cheese-making industry?
Produced from bacteria: high productivity yield, better texture, reduced bitterness
Which enzymes are involved in high fructose corn syrup production?
alpha amylase, amyloglucosidase, xylose isomerase
How can a protease be used to build dipeptides?
in low water conditions, equilibrium of peptide bond hydrolysis is reversed, favouring production of peptide bonds
What are biosensors?
electronic analytical devices which are used for monitoring blood glucose and cholesterol levels. based on enzyme action
how does a biosensor work?
target molecule binds to an immobilised enzyme (specific), which sends a signal to the transducer to detector
how is a signal detected in a biosensor?
by production of H2O2, which reacts with dye to produce a colour change (urine dipsticks), or electrical current recorded by reduction of H2O2
What are microelectrode biosensors?
immobilised enzymes react with analyte, which produces H2O2, which is oxidised by Pt electrode, converting it into an electrical signal
