L6-8: Intro to Enzymes I, II & III Flashcards
What are activating subunits?
- these are components of a holoenzyme that are required for the enzyme’s (of which they are associated) catalytic activity
Draw the Lineweaver-Burk plot and indicate what each intercept, slope and axes mean. Indicate what navigating around the graph means.
What are two common competitive inhibitor drugs used in everyday life?
- Acetaminophen and advil. These are prostaglandin synthase inhibitors.
Do enzymes change the free energy of the substrates and / or products of a reaction?
- No, delta G is unchanged.
An enzyme has a histidine residue that is essential for catalysis. This enzyme is inactive a pH < 4 and pH > 9. Why?
- Histidine residues have a pKa of approximately 6.0, which at physiological pH means that they can exist in protonated or deprotonated form. When at pH of 4, residue has strong tendency to exist in protonated form (acid) and cannot act as a good proton donor. When at pH of 9, residue has strong tendency to exist in deprotonate form (base) and cannot act as a good proton acceptor. Therefore, catalysis relying on acid/base interactions between enzyme and substrate no longer occur and function is lost. All residues have optimum pH range in which they work to enhance reaction rates, extremes and out of this optimum have decreased or negligible reaction rates.
How can one experimentally distinguish between noncompetitive and irreversible inhibition?
- removal of irreversible inhibitor does not restore enzyme activity; however, removal of non-competitive inhibitor will restore enzyme activity.
How does aspirin function?
- It is an irreversible inhibitor of prostaglandin synthase and binds to the active site of the enzyme forming a covalent complex.
Define cofactors.
- Cofactors are defined as small molecules that are required for activity of enzymes. They include metals (metaloenzymes) and small organic molecules (coenzymes).
The Km for a substrate often corresponds closely to metabolic concentrations of the substrate. Why is this advantageous?
- When changes to substrate concentration occur, enzyme is sensitive enough to decrease reaction rate or increase reaction rate.
Late onset hyperammonemia results in elevated ammonia in the blood and is caused by a mutation to an arginine residue to be replaced by a glutamine residue. As a result, Km is 60 fold higher than the normal enzyme. Explain how hyperammonemia is occurring.
- Since the mutated protein has a Km that is 60 fold higher than the normal protein, this means that the protein has low affinity for the substrate. At physiological substrate concentrations, reaction rates are now lower due to decreased enzyme-substrate interactions (ez activity) and necessary down stream reactions that serve to “rid” the body of excess ammonia and hindered.
Explain what an irreversible inhibitor is?
- An irreversible inhibitor is one that forms covalent complexes with active site residues in enzymes, chemically modifying and therefore inactivating an enzyme. No change occurs with increased substrate concentration, nor removal of inhibitor.
What are active sites? Where are active sites often located? Why?
- Binding sites refer to small areas in an enzyme (relative to total enzyme structure) that bind substrate. They are often in a pocket, crevice or cleft in the enzyme where water is excluded (unless participating in reaction). They need to be discrete and hidden to prevent cross reactions / side reactions from occurring – specificity is key.
What are suicide substrates?
- Suicide substrates, aka Trojan Horse substrates, are special classes of irreversible inhibitors that only become inhibitors through the catalytic action of the target enzyme. They bind covalently at active site residues.
Describe how enzymes can react with chiral substrates in a stereospecific manner.
- In order for an enzyme to bind a chiral substrate, there needs to be 3 points of interaction between the substrate and the enzyme. For this purpose, if only two atoms branched from a chiral center interact with the enzyme, but not the 3rd because the substrate is of a different stereochemistry, a reaction will not occur. Needs 3 point interaction. This allows for stereospecific binding and reactions.
How do you convert the rectangular hyperbolic V vs. [S] curve into a straight line using the Lineweaver-Burk (double-reciprocal) method. What are the X- and Y-intercepts and the slopes of this plot? How could the Vmax and Km be determined using this plot?
- 1/v = (Km/[S]).(1/Vmax) + 1/Vmax (y = mx + b form) - The x intercept = -1/Km – the negative reciprocal gives you Km - The y-intercept = 1/Vmax – the reciprocal gives you Vmax - Slope = Km/Vmax
How do many drugs work against enzymes?
- Many drugs reduce the rate of ez catalyzed reactions. Most drugs are reversible, competitive inhibitors, meaning they compete with substrate for binding at the active site.
Why are non-competitive inhibitors potentially better drugs than competitive inhibitors? What is the difficulty in manufacturing them?
- Non-competitive inhibitors would perhaps make better drugs since they can inhibit enzyme activity irrespective of the substrate concentration. Would not have to worry about dosing based on substrate concentration. They are difficult to manufacture from the design perspective. Since non-competitive inhibitors bind in a location other than the active site, yet upon binding cause active site to be unable to bind substrate, how can you determine where this other site is?
Define coenzymes.
- Coenzymes are cofactors that are small organic molecules. Coenzymes are typically derived from vitamins. Eg. B1, niacin, B6, B2 etc.
Describe how the determination of the 3-D structures of the isozymes of prostaglandin synthase aided in the design of drugs that selectively inhibit COX-2.
- NSAIDs act by inhibiting prostaglandin synthase, which are responsible for pain and inflammation. This enzyme has two enzymatic activities, one of which is cyclooxygenase activity and the other, hydroperoxidase. NSIADs inhibit the cyclooxygenase. Turns out there are two isozymes for this enzyme, COX-1 and 2. - COX-1 protects gastrointestinal mucosa, while COX-2 is associated with inflammation of arthritis. - As a result of this discovery, drugs have been designed specifically as inhibitors to COX-2. Eg. Celebrex, bextra and vioxx.
What is product inhibition? Give an example of an enzyme that is regulated by product inhibition.
- Refers to the product of an enzyme catalyzed reaction inhibiting the enzyme that made it when concentrations of itself are high. - Example: G6P inhibits hexokinase
What are the advantages of a multienzyme/multifunctional protein complex?
- Elimination of substrate diffusion/dissociation between sequential enzymatic reactions. - Coordinated control of sequence enzymatic steps. - Coordinated, stoichiometric gene expression of enzymatic activities.
What is the relationship between the magnitude of Km and enzyme/substrate affinity?
- inverse relationship - low Km = high S-E affinity - high Km = low S-E affinity - means some enzyme-substrates interact better than others
Describe and explain graphically with 1/V vs. 1/[S] plots and pictorially with drawings: • competitive inhibition
Describe and explain graphically with 1/V vs. 1/[S] plots and pictorially with drawings: • noncompetitive inhibition
Draw a substrate saturation curve and indicate Vmax, Km, Vmax/2.
Draw the Michaelis-Menten saturation curve for unhibited enzyme, competitively inhibited enzyme, non-competitively / irreversibly inhibited enzyme. Indicate what occurs to Km and Vmax as a result of inhibition when compared to uninhibited.
