Biocatalysis: Science and Technology

Vakinformatie

Voor 2021 heette het vak Advanced Enzymology. Examen is volledig mondeling. Vragen schriftelijk voorbereiden en dan alles mondeling uitleggen

ECTS-fiche: https://onderwijsaanbod.kuleuven.be/syllabi/e/G0U19AE.htm

Examenvragen

9/01/2026

Question 1

Figure of a kinetic profile of an NADH consuming reaction (fig 3.29) (enzyme that uses substrate and cofactor NADH).

1. Propose and draw an experimental setup to obtain this data (Stopped flow...)?
2. Why does this enzyme need NADH?
3. Give a possible reaction scheme of the reaction + rate equations, explain
4. How could you get a concentration profile (develop kinetic model) quantitatively?
5. Bijvraag: What are the four different ways of enzymes to catalyze reactions?

Question 2

Figure of the four basic approaches that can be used in metabolic engineering (fig 5.2).

1. Explain the different approaches
2. What is enzyme activity, how it is determined?
3. Why is it possible to tune the activity of enzymes?

10/01/2025

Question 1

Figure of reaction mechanism of chymotrypsin (fig 2.13).

1. What does this figure tell us about the different mechanisms that enzymes use to accelerate

reactions? Explain.

1. Are any mechanisms not shown in this figure?
2. Should we expand this view when considering the figure below? (fig 11.14 The interconnection among protein structure, dynamics, and function, convential and emerging view). What is meant with this?

Question 2

The figure below has been reproduced from the course materials (fig 4.1, Stochastic chemical kinetics). It shows the amount of X remaining during a reaction occurring via a simple reaction scheme X → Y. You can assume that this is an elementary reaction. a) What is an ‘elementary reaction’? b) Write the conventional kinetic equation describing this reaction. Solve it analytically. c) What are the red and blue lines in the figure? Why are they jagged (not smooth)? d) Why would we want to carry out experiments under conditions such as those in this figure? What is the advantage? What is the drawback? e) What kind of methods could in principle be used to carry out such experiments?

Question 3

The figure below has been reproduced from the course materials (fig 6.3). a) Discuss the different steps. Why are they needed? b) How could one obtain an enzyme that functions better for a particular purpose?

27/01/2022

Question 1

Figure of Alphafold improvement in CASP and myoglobin structures.

1. What drives protein folding? Which mechanisms are involved?
2. Why do we say that the structure of a protein is encoded in the primary sequence?
3. How do AlphaFold and related algorithms work? (not a detailed description, just generally)

Question 2

Why do proteins need cofactors? Explain with 1 or more examples.

Question 3

Figure of Ohno and IAD model is given.

1. What does neofunctionalization mean in the scheme below?
2. What does specialization mean?
3. Why does the IAD model, which is thought to be more correct than the Ohno model, indicate that neofunctionalization happens first?
4. What does this mean for the specificity of natural enzymes?

9/01/2021

Question 1

Figure below shows a 1,4-elimination of a carboxylic acid catalyzed by an enzyme.

1. What do ‘B’ and ‘M+’ indicate?
2. Indicate in the figure which groups are part of the enzyme, and which are part of the substrate
3. How is this reaction accelerated by the enzyme? Try to explain in your own words how the enzyme can make this reaction go faster. Alternatively, you can explain why the reaction would be slower in just an aqueous solution (that is, a solution without enzyme).
4. Would it be easy to perform similar catalytic steps by adding small organic molecules to a solution? Why (not)?

Question 2

We have seen that reaction rates of an enzyme presented with two competing substrates are given by: v/v' = {(kcat/KM)/[S]}/{(k'cat/K'M)/[S']}

1. Give an intuitive explanation for kcat and KM. What are they?
2. What determines kcat and KM? What limits how well an enzyme can distinguish between two substrates?
3. How might an enzyme be able to obtain a higher specificity than is predicted by this equation? Is there a cost to be paid?

Question 3

Graphs of 2 enzymes from different organisms with 2 different temperature dependence (Mesophilic and thermophiles). X is the fraction of unexchanged hydrogen atoms, the x-axis represents time.

1. What is ‘H/D exchange’ and what are we trying to measure from this?
2. What can we learn from this?

24/01/2019

PART 1. Oral examination by prof. Peter Dedecker

1. Given a kinetic scheme with product feedback, compose the differential equations that govern this system.
2. Explain the value of k' = k1 (1+([S2]/K)^n) [S1].
3. Explain the concept and the procedure of numerical integration for a simulation of a product feedback scheme.
4. Given data of real, in vitro experiment of [Ca2+]i by Fura-2 and [cAMP] by IUCP, list the problems that may arise in such an experiment and how to surmount them. How does an experiment like this work practically?

PART 2. Written examination by Dr. Fabian Hertel

1. Sketch an acid-base, electrophilic or nucleophilic catalysis pathway using a correct amino acid of choice.
2. Give the correct action mechanism for two different amino acids.
3. What role do metal ions play in enzymatic catalysis?
4. Sketch a 2D folding funnel. Mark where the completely folded enzyme, and a unfolded intermediate are located. What mechanisms exist in nature to make the folding process more efficient?
5. Your research requires an enzyme that is capable of converting MDMA to the non-pyschoactive compound benzaldehyde. There exists a catalytic antibody that is capable of this reaction. How do these catalytic Abs work? Is further optimisation necessary? Describe one method to design an artificial enzyme.