Enzyme catalysis

L1 Define Bronsted acids and bases!

L1 Define Lewis acids and bases and compare this to the Bronsted concept!

L1 Define the terms nucleophile and electrophile!

L1 Describe the mechanism of electrophilic addition of acid-catalyzed hydration of 2-methyl- propene!

L1 Compare the two major nucleophilic substitution reactions regarding formed intermediates and the type of substrates!

L1 Name the two major groups of compounds bearing a carbonyl group and compare their reactivities!

L1 Order the following compounds according to their reactivity starting with the most reactive species: thioester, amide, acyl phosphate and ester! Draw the functional group for each compound!

L1 Name three mechanisms of elimination reactions and draw one of those explicitly!

L1 Which proteinogenic amino acids exhibit predominantly a charged side-chain at pH 7?

L1 Which amino acids occurs most frequently in the active site of enzymes? Rationalize why this might be the case.

L1 Explain why cysteine is the most commonly occurring amino acid in covalent catalysis.

L2 Name common principles and differences between chemical and enzyme catalysts!

L2 Name five different chemical mechanisms of enzymes!

L2 Draw the reaction coordinate of an enzymatic reaction going from substrate to product via two intermediates! Indicate the free energy change of the reaction and the activation energy for the enzymatic reaction in comparison to the uncatalyzed reaction!

L2 What is the “orientation effect” in enzyme catalysis and provide an estimate for how large the rate enhancement can be in principle for an enzyme by properly orienting substrates?

L2 Describe an example where an enzyme physically distorts its substrate with an emphasis on bond activation and energy of the substrate!

L2 Describe the principle of ground-state destabilization and transition-state stabilization and provide an example for each!

L2 Describe the key steps and involved intermediates of the reaction mechanism of serine proteases!

L2 Compare a normal (ionic) hydrogen-bond with a low-barrier hydrogen bond and a single-well hydrogen bond! Name an enzyme where a low-barrier hydrogen bond is thought to be catalytically relevant!

L2 Detail the currently accepted mechanism of lysozyme action!

L2 Name four different classes of proteases including one example for each and draw the mechanism for two families! Indicate where enzyme magics is required to catalyze chemically difficult steps!

L2 Compare proteases and esterases regarding their active site machinery and explain the differences!

L2 Draw the mechanism of acetylcholine esterase! Name two classes of acetylcholine esterase inhibitors and explain how they function!

L3 Describe briefly the mechanism of phosphomutases!

L3 Explain the term ‘isomer’! What types of isomerism are known? Derive a scheme for all types of isomerism. Provide examples (molecules or pairs of molecules) for the different types of isomerism!

L3 Explain two different mechanisms of epimerases acting on sugar substrates!

L3 Name three different enzymatic reaction mechanisms used by racemases! Name one example each and name commonalities and differences between the three mechanisms?

L3 Radical isomerases: name the two radical initiators cofactors including the mechanism of radical formation! Name one enzyme for each cofactor!

L3 Explain the two possible chemical routes for isomerization between aldoses and ketoses! Name one example each for an aldose-ketose isomerase using this mechanism! Name experimental strategies to distinguish between the two alternative mechanisms for a given isomerase! Which enzymatic reaction steps are particularly difficult to catalyze?

L4 Explain possible chemical routes (4) to afford a decarboxylation of metabolites?

L4 Compare thiamin diphosphate-dependent and PLP-dependent decarboxylases! Name commonalities and differences in the mechanisms! Describe how PLP enzymes control reaction specificity by stereoelectronic control!

L4 Explain the mechanism of pyruvoyl-dependent decarboxylations!

L4 Which reaction is catalyzed by acetoacetate decarboxylase? Draw the mechanism and explain the different functional roles of the two neighboring lysine residues!

L4 Explain the catalytic strategy employed by mevalonate pyrophosphate decarboxylase!

L4 Name different suggested mechanisms for oritidine-5’-monophosphate decarboxylase! Which one(s) are supported or rules out by the experimental results?

L4 Explain the mechanism of pyruvate formate lyase (PFL) and of the required PFL activase!

L5 Draw the chemical structure of the nicotinamide ring of NAD(P) + in the oxidized and reduced NAD(P)H state!

L5 Draw the mechanism of hydride transfer in a NAD+ -dependent dehydrogenase that interconverts alcohols and ketones!

L5 Is hydride transfer in NAD+ -dependent dehydrogenases stereospecific? Explain the underlying mechanism and suggest an experiment that can prove or rule out stereospecific hydride transfer!

L5 Draw the chemical structure of thiamin diphosphate and mark the reactive atom! Why is that particular atom so reactive?

L5 Draw the chemical structures of substrate classes of thiamin enzymes and indicate which bonds are susceptible to cleavage by thiamin!

L5 Draw the chemical structure of the central carbanion-enamine intermediate in thiamin enzymes and name two possibilities how reaction can progress from this state!

L5 Draw mesomeric structures of the alpha-carbanion derived upon PLP-assisted decarboxylation!

L5 Draw the chemical structure of PLP in covalent linkage with an alpha-amino acid for a PLP i) decarboxylase and ii) racemase!

L5 Draw the isoalloxazine ring system of flavin cofactors in the oxidized and two-electron reduced state!

L5 Name the two half-reactions typically catalyzed by flavoenzymes and provide an example!

L5 Draw the structure of the biotin cofactor in resting and “cargo-loaded” state! Explain how cargo (substrate) is chemically activated in biotin enzymes?

L5 Draw the chemical structure of the pyruvoyl “cofactor” and name a reaction where this cofactor is acting!

L5 What are quinoproteins? Name two classes of these and the reactions they are typically catalyzing!

L6 Write the Michaelis-Menten equation and draw the corresponding v-S plot highlighting Vmax and KM!

L6 Explain the terms and provide units for catalytic constant, Michaelis constant and catalytic efficiency!

L6 Write the Michaelis-Menten equation of an enzyme displaying positive cooperativity and draw an associated v_S plot! Name a method to estimate the Hill coefficient! What is the Hill coefficient?

L6 Name different types of bisubstrate enzyme reactions! How can these different mechanisms be distinguished by appropriate kinetic experiments?

L6 Explain how a competitive inhibitor is acting! Draw the corresponding v_S plot and the Dixon plot! Which kinetic constants are affected by a competitive inhibitor? Write the Michaelis-Menten equation for an enzyme reaction that is inhibited by a competitive inhibitor!

L6 Explain how a non-competitive inhibitor is acting! Draw the corresponding v-S plot and the Dixon plot! Which kinetic constants are affected by a non-competitive inhibitor? Write the Michaelis-Menten equation for an enzyme reaction that is inhibited by a non-competitive inhibitor!

L6 Explain how a uncompetitive inhibitor is acting! Draw the corresponding v-S plot and the Dixon plot! Which kinetic constants are affected by a uncompetitive inhibitor? Write the Michaelis-Menten equation for an enzyme reaction that is inhibited by a uncompetitive inhibitor!

L7 Draw the reaction profile for an enzyme-catalyzed reaction and indicate the pre-steady-state and steady-state phases! Highlight the concentration profile for E (free enzyme), ES (enzyme-substrate complex), S (substrate) and P (product)!

L7 Name three different methods used for pre-steady-state analysis of enzyme reactions! Name the time regime that can be analyzed with each method, the type of reaction amenable for analysis and the signals/readout used! Explain one method in greater detail!

L7 Write the rate law for a first-order reaction and draw the corresponding progress curve!

L8 How is protein homeostasis maintained in eukaryotes?

L8 Name two major protein degradation pathways in eukaryotes.

L8 Which cellular systems monitor protein folding?

L8 Name abundant AAA ATPase family proteins and their functions.

L8 Which amino acids are included in the pore-loop that interacts with extended polypeptides?

L8 Outline the mechanism by which AAA+ ATPases unfold substrates.

L8 What is the number of ATPase subunits required to constitute a proteasome ATPase motor?

L8 Name three peptidase activities of the proteasome.

L9 For each of the enzyme classes (E1, E2, E3, and DUB) list the reaction educts and products.

For each enzyme class, list the names of the chemical reactions that are catalyzed by it.

L9 Define and draw the structural formula of an amide bond, peptide bond, and isopeptide bond?

Draw by a combination of cartoon (protein elements) and formula (decisive chemical bonds) a substrate protein that is ubiquitinated at a serine residue and at a lysine residue.

L9 Describe in what sense the E1-catalyzed reaction resembles a step during protein synthesis. What is the common principle that renders both reactions irreversible?

L9 Can you think of why the ubiquitination system may have evolved as a catalytic cascade (rather than single enzymes catalyzing ubiquitination)?

L9 Ubiquitination sites on cellular proteins are typically identified by a trpysin-mediated digest of the sample followed by mass spectrometry. Use a cartoon representation of a ubiquitinated substrate to explain schematically which ubiquitin-derived digestion products are searched for.

Hint: You need to look up the amino acid sequence preference of trypsin and the amino acid sequence of ubiquitin.

L9 What are specific challenges in developing PROTACs for therapeutic applications? Consider the molecule have to have, its dosage, and requirements with regard to the target protein and ligase?

L9 If you had the opportunity to develop drugs against ubiquitin-system enzymes, which family would you choose and why?

L9 Do you expect the ubiquitin system will furnish as many drugs as protein kinases and why? What are the fundamental differences between these groups of targets in terms of drug discovery?

L10 Use the simplified Eyring equation:

k = kBT/H * exp(-deltaG / RT),

with kB as the Boltzmann constant, h the Planck constant and R the gas constant, to derive the predicted energy barriers for reactions with a k=1000 s-1, 1 s-1 and 0.001 s-1 at 300 K. Compare these energies and their differences to the strength of a hydrogen bond between two water molecules (~20 kJ/mol).

L10 Discuss the differences between a “real” bond potential and a model harmonic potential.

L10 Why might it be difficult to computationally model metalloenzymes with molecular mechanics?

L10 For many systems, it has been postulated that the origin of catalysis comes from electrostatic transition state stabilization. Choose one example from previous lectures and discuss how this stabilization is specific to the TS. Can you find one or more reasons why this should be a particularly dominant feature in enzymatic catalysis?

L10 What experimental approaches could one use to assess whether electrostatic stabilization is the main catalysis factor? (you may again pick a system to discuss)

L10 Consider the following scenarios. In which cases would you deem simple MM studies applicable and in which cases do you believe QM/MM is required?

a) diffusion-controlled reactions

b) proton transfer reactions

c) elongated bonds/structural stress

d) stereoselectivity imposed through steric shielding

e) radical reactions

L10 Give an example of an experimental method to track intermediates of an enzymatic reaction, and list its limitations.

About 40 different tRNAs are sufficient to decode 61 sense codons. Why is this possible and what is the molecular basis for this phenomenon?

Explain how the high speed and accuracy in translation is achieved. How long does it take to synthesize an average E. coli protein (333 amino acids)? Assuming an error frequency of 3x10-3, what is the probability that it is synthesized with a mistakes?

In an elegant experiment performed in 1962, a cysteine that was already attached to tRNACys was chemically converted to an alanine. These alanyl-tRNACys molecules were then added to a cell-free translation system from which the normal cysteinyl-tRNACys molecules had been removed. When the resulting protein was analyzed, it was found that alanine had been inserted at every point in the protein chain where cysteine was supposed to be. Discuss what that experiment tells you about the role of aminoacyl-tRNA synthetases and the ribosome during normal translation of the genetic code.

How is the energy of ATP and GTP hydrolysis utilized during translation? How is ATP/GTP consumption coupled to acceleration of protein synthesis? Which of the translation factors are GTPases? What is the role of GTP hydrolysis in translation?

How do aa-tRNA synthetases recognize the proper tRNA and amino acid? How do they correct errors of amino acid recognition? Isoleucine and Valine are almost identical. They differ by one CH2 group. How do isoleucyl tRNA and valyl-tRNA synthetases discriminate between the two amino acids? The extra methyl group of isoleucine could be expected to provide -2 to -3 kcal/mol of free energy in binding; hence 1/100 would be the error frequency. What is the observed error frequency and how is it achieved?

In an in vitro translation system from Staphylococcus aureus, the model mRNA directs for synthesis of a protein that starts with amino acid sequence fMet-Phe-Thr-Ile… Three new antibiotics isolated by a company you are now working for inhibit the translation of the mRNA in a different way. Compound RI-333 inhibits synthesis of peptides that are longer than fMetPhe; formation of fMetPhe is not affected however. In contrast, compound RI-506 allows for synthesis of peptides that are 6-7 amino acids long, after which the synthesis is stalled. Finally, with the third compound, RI-700, many oligopeptides of different length are synthesized, but a large part of them does not have an expected amino acid composition. What can you suggest as to the mode of action of these antibiotics? How would you test your suggestions? What are the following steps to further develop these drug leads? What are the structural and functional differences between prokaryotic and eukaryotic ribosomes? What about mitochondrial ribosomes?

Which chemical reactions are catalyzed by the ribosome?

Which part of the ribosome catalyzes the peptide bond formation - the RNA or the protein? How would you address this question experimentally?

The ribosome is a molecular machine. Explain why.

Is the genetic code identical in all organisms? Is recoding and redefinition the same thing? Provide examples. Why is recoding used?

The company where you ordered the plasmid encoding for a mammalian gene for expression in E. coli suggests implementing the “optimal codon usage” for the coding region. What is that? Will you choose this option? You made a choice and tried to express the protein, but it does not express well in E. coli. Why do you think this might happen? What would you suggest to improve the expression level?

What are the main steps in prokaryotes and eukaryotes initiation? Name some main differences.