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2. BIOC 2306 S1 > Enzymology P2 > Flashcards

Enzymology P2 Flashcards

1
Q

What is rational protein engineering?

A

Synthesis - new enzymes, designer proteins, new proteins, improved enzymes and engineered antibodies (e.g. to fight cancer cells)
Initially worked well, but subsequent injections lead to a immune response fighting the antibodies (as they were foreign)

Analysis - to understand enzyme function, recognition and structure/activity relationships

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2
Q

How can we ‘improve’ enzymes?

A

Improve thermostability, pH optimum and substrate specificity etc…
This depends on what we define as ‘improved’ conditions

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3
Q

How can we assess an improved enzyme?

A

Assess substrate specificity:
Substitute Kcat into Michealis Menten equation
v = Kcat[E][S]/(Km + [S])

At equal [E] when [A]=[B]
(Va/Vb) = (Kcat/Km)a / (Kcat/Km)b
The ratio will tell us how well the enzyme is working to see if we have improved the enzyme

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4
Q

Describe an early example of enzyme engineering?

A

Engineered Trypsin: normally cleaves next to a lysine, but now cleaves a glutamate
This new enzyme didn’t have any activity
Therefore it is much easier to destroy activity than it is to create activity

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5
Q

How would we engineer an enzyme - give an example?

A

In order to engineer an enzyme we need to understand everything about the enzyme

Example: Lactate dehydrogenase
We want to change the substrate pyruvate to oxaloacetate so the dehydrogenase works with that instead of pyruvate
New substrate is bigger and more negative

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6
Q

What factors did Prof JJ Holbrook in Bristol look at to engineer an enzyme?

A

Overall active site charge balance
Influence of substrate and active site volumes
Direct electrostatic complementarity

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7
Q

Describe alteration of LDH in relation to: overall active site charge balance?

A

This charge balance is just in the active site

They chose 2 mutations:
Asp197 to Asn - charge buried in protein
Greater change

Glu107 to Gln - charge exposed to solvent
Limited change

To see if any change were made we can plot Log Kcat/Km of the substrates
Effect is more pronounced if charge is buried

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8
Q

Describe alteration of LDH in relation to: influence of substrate and active site volumes?

A

Thr246 to Glycine - T246G
This leads to a bigger active site
This gave a switch in specificity from pyruvate to oxaloacetate
The active site is now too big for pyruvate - therefore can allow water molecules in also, interrupting the mechanism

Didn’t make a huge overall difference

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9
Q

Describe alteration of LDH in relation to: direct electrostatic complementarity?

A

Gln102 to Arg - Q102R

This is very successful - increases specificity for oxaloacetate 10^6 = 10^3 increase
Now pyruvate is 10^3 specificity which is a 10^4 decrease

Direct electrostatic complementarity is most important

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10
Q

How do we make alterations of LDH away from a natural substrate?

A

Branched substrates for LDH don’t function well at all
We need to introduce more flexible space into active site

We do this by trying to make the JAW and loop more flexible and hydrophobic e.g.
More flexible - A235G, A236G
More hydrophobic - Q102M, K103V, P105S
Or do both

We find that engineering proteins we can’t just do a single amino acid swap - we need combinations of mutations
= synergistic mutations

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11
Q

How else can we alter an enzyme’s function if not interaction with substrate?

A

We can alter the interaction with the co-factor

e.g. Glutathione reductase and NADPH

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12
Q

Describe glutathione reductase?

A

This catalyses the redox of glutathione
This reduces the di-sulfide bond = reduced glutathione
This is being used as an example as we are engineering the specificity of the cofactor

Dimeric enzyme
Contains FAD
Contains a redox active disulphide bridge

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13
Q

What is the mechanism of glutathione reductase?

A

NADPH binds in the active site next to the FAD
FAD effectively divides the active site into two parts
One has the redox active site where glutathione binds and the other where NADPH binds

The electrons in the charge transfer complex can be used to reform the disulphide bridge

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14
Q

How/why are we focusing on changing the NADPH cofactor affinity?

A

The only difference between NADH and NADPH is a phosphate group

The active site/pocket for NADP+ is formed with 2 Arginine residues (very positive)
They electrostatically bind the negative phosphate
The pocket is even more positive as it contains His and Lys relatively close to the active site surface

We may want to change the enzymes cofactor as for example NADH is a lot cheaper than using NADPH

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15
Q

How can we approach altering cofactor activity in glutathione reductase?

A

As we are dealing with a cofactor there are a lot of enzymes that bind NADPH
We can take a more bioinformatic approach

Positive pocket for NADPH
Conserved motifs

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16
Q

Describe alteration of glutathione reductase in relation to: the positive pocket for the cofactor?

A

Alter the postive amino acid residues from arginine to leucine and methionine

We knocked out the NADPH activity but didn’t increase NADH activity - destroyed activity

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17
Q

Describe alteration of glutathione reductase in relation to: conserved motifs?

A

NADH/NADPH amino acid residues are highly conserved across organisms
It seems they had a pattern
NADPH
GxGxxAxxxA

NADH
GxGxxGxxxG

Therefore altering alanine to glycine
After many synergistic changes - this increased the activity of NADH greatly

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18
Q

How can we measure the switch in specificity?

A

(Kcat/Km)desired / (Kcat/Km)natural

Mutant/Wild type
The equation above on both lines

For glutathione reductase alterations = 17,700 fold specificity change

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19
Q

Describe the changes from the original motif of glutathione reductase to the mutated motif?

A

We mutate Ala179 to glycine
The methyl group can move out of the way so the 175 residue swings around making more room for the glutamine residue at 197 to making interaction with the 2 OH’s rather than with the phosphate on the NADPH

20
Q

How could we design an enzyme from scratch?

A

There would be significant difficulties – the protein folding problem
We could use pre-existing protein scaffolds and build in activity
Effectively based on Transition State Theory – we know that the transition state is bound the best

Computational enzyme design
Catalytic approach

Could do both to see if the outcome has the desired activity

21
Q

Describe the computational enzyme design approach?

A

Design in 3D the require transition state - on the computer
Design in 3D a complementary active site - on the computer
= theozyme (theoretical enzyme)

Find structures that match shape, with residues in the position of R1, R2 and R3 in the correct orientation around the transition state
There could be hundreds of structures
Optimise amino acid side chains to be complementary to transition state

22
Q

Describe the catalytic antibody approach?

A

Make an analogue of the transition state - antibodies raised against and this will bind the transition state and should be catalytically active

23
Q

What is directed evolution in relation to natural evolution?

A

Natural evolution: Random genetic mutations occur within the DNA of an organism and useful mutations are retained as they enhance survival

Directed evolution mimics natural evolution but at the level of a single gene in the laboratory, to create mutated genes that can selected for their improved properties (usually due to the better protein/enzyme encoded)

Both involve the introduction of RANDOM mutations and the selection of improvements

24
Q

How does the sequence space affect directed evolution?

A

If we created all protein combinations from a tiny 60 aa sequence - the weight would be X10^30 larger than the earth

Therefore we use ‘sparse sampling’ used in directed evolution experiments
We use a 1.5ml tube
This samples a very small part of the complete library, but with enough diversity to select interesting proteins

25
Q

What is the usefulness of directed evolution?

A

Improve protein properties or identify new functions e.g.
pH thermal and/or stability
pH and/or temperature optima
binding properties - altered Km for substrate(s)
catalytic properties - enhanced Kcat
selection of altered fluorescence properties
selection of new binding proteins

26
Q

What is the iterative cycle of directed evolution?

A

Take the gene(s) - don’t need to know anything about it, as we are just looking for mutants

Introduce diversity - SM, EP-PCR, DNA shuffling, Family DNA shuffling, StEP, RACHITT, ITCHY, ADP, SCHEMA

Express many variants in a suitable host (E.coli)

Identify suitable variants - chromogenic/fluorogenic substrates, coupled enzyme assays, IVC, Surrogate substrates, growth selections

Repeat
Until - final improved enzyme is found

27
Q

How does directed evolution work?

A

We have to be able to physically have the code of the gene for the random variant we selected in order to move onto the next cycle = phenotype-genotype link
Phenotype-Genotype link – need to be able to recover the DNA encoding the improved protein

Introduce random mutations into the DNA so do not need to decide what mutations to make
Error-prone PCR, DNA shuffling

Create a library in which each gene copy contains a few mutations

Set up a way of screening or selecting improved proteins encoded by the library

28
Q

How do we establish a phenotype/genotype link?

A

In vivo - cell transfection
Most common is to use a cell - as it contains nucleic acid we have manipulated and it expresses the protein
This restricts the library size to 10^10 due to transfection efficiency

In vitro - no transfection step = larger libraries 10^14
Technically demanding
The coding sequence must be trapped in a complex with the protein that it encodes

29
Q

What are some different ways to establish phenotype-genotype links?

A

Phage link

Yeast or bacterial cell
Add a plasmid containing out gene - this cell expresses the protein
Or the plasmid can be encoded so the proteins are displayed on cell surface

Ribosome display
Technically demanding method
Stall the complex where the ribosome doesn’t fall of the expressed protein - has a peptide linked between the two

mRNA
The stalled ribosome has made this, forming an artificial covalent link between the peptide and mRNA
Also very technically demanding - very highly levels of variants

Cis-DNA

Oil/water droplets

30
Q

Describe the phenotype-genotype link of oil/water droplets?

A

if we take a water solution containing DNA and a cell extract
The DNA can be made into mRNA and proteins - as the water solution will take the DNA to make proteins
If we put the water solution into oil we get drops of water within the emulsion
Each drop can be modified so it only contains one molecule of water - where each drop contains one DNA molecule and therefore one protein
They can work at very high rates - 1000 sec-1

31
Q

Describe the phenotype-genotype link of phage display?

A

Using a bacteriophage - most common M13
M13 - single strand DNA genome, including any inserted coding region, is packaged in the phage particle
It is encoded by proteins forming a coat

Proteins can be expressed as translational fusions to coat proteins
We can encode our proteins that we want to express into these coats - displayed on the coat
major coat protein gp8; 50aa, 3,000 copies – good for peptides
minor coat protein gp3; 406aa; 5 copies – good for proteins

32
Q

How do we introduce diversity into the protein?

A

Error prone PCR
DNA shuffling
Family shuffling

33
Q

Describe error prone PCR?

A 
Reduce PCR fidelity
Increased conc. Taq, MgCl2
Increased extension time
Altered nucleotide concentration
dATP & dGTP 0.2mM dCTP & dTTP 1mM

We WANT to make mistakes to force the copying process to make more mutagens
Mutazyme II is a blend of Mutazyme I DNA polymerase and a novel Taq DNA polymerase mutant that exhibits increased mis-insertion and misextension frequencies

The single amino acid changes are limited to 5 other amino acids + stop codon
= limited by codon usage

34
Q

Describe DNA shuffling?

A

Error prone PCR is combined with a recombinant method = DNA shuffling
From Error prone PCR - produce fragments (using sonication/DNase)
Denature them and use a PCR like reaction to anneal and extend through self-priming
The sequence is then very shuffled/mixed up

Population variation through recombination

35
Q

Describe family shuffling?

A

DNA shuffling of a FAMILY of sequences should recombine these to generate new variants
A high proportion of the products should be functional (folded, stable, display activity)
Inactive variants will still exist but should be minority

Example - used for biological washing powder methods (serine proteases)

36
Q

What is the first law of directed evolution?

A

You get what you screen for

In practice we can select or screen

37
Q

Describe selection?

A

Selection is ideal if a survival phenotype is associated with protein e.g. Growth v no growth in antibiotic resistance

We grow the library to see this - if they live they have the enzyme we are looking for
But it can be difficult to devise a live/dead selection for many proteins that we might want to evolve

It can be used if there is an obvious phenotype in a plate assay = coloured products or a coupled assay of an enzyme with another enzyme system that produces a colour

If nothing obvious available we have to screen

38
Q

Describe screening?

A

Many proteins have no obvious phenotype for bacterial growth or colour formation in a plate based assay
Need to screen individual clones for activity; 10^3 -10^5
Need to be careful not to over-mutate - most clones should show activity

Enhanced throughput by using robotics but still limited to numbers that can be screened and expense
Very inefficient and expensive if most variants are inactive/unfolded so need to ensure relatively low mutation rate often 1-3 aa per gene
Has driven the need to generate smaller more focused libraries

39
Q

Describe directed evolution of fungal peroxidase?

A

Used error prone PCR to mutate the WT gene
2 clones with improved residual activity
T34A, S198T, E239G
I195V, E239V

Higher residual activity by mutating E239
Even after solving the 3D structure this would not have been identified without a random mutagenesis/screen strategy

40
Q

Describe direction evolution of the GFP protein?

A

It is useful to have different colours of the protein
We can do directed evolution with GFP
All we have to do is look at the fluorescence of the bacterial colonies

41
Q

How do we tackle using a reduced sequence space?

A

CASTing - (Combinatorial Active site Saturation Testing)

We take ‘groups’ of the wild type and screen them
We then take the best of the group and then repeat - screening further
This isn’t perfect as some may need certain combinations that we didn’t try in the initial stages

42
Q

What element other than substrate binding can affect directed evolution?

A

Stereoisomers

Different stereo-isomers have different biological effects e.g. Different smell
We want to use directed evolution to change the stereochemistry within the active site - this is hard as it is only slightly altering the shape
We can use directed evolution as we don’t need to understand it fully

Enantiomers = mirror images
If we have 2 or more ‘chiral’ centers = diastereoisomers

43
Q

Describe how we carry out genetic selection of enantioselective variants?

A

Through 6 generations of directed evolution we can see an increase in preference to a certain enantiomer - altering an amino acid residue each time (mutation)

The first mutation is maintained for the 2nd generation, and each mutation after contains the previous mutation as that type is better - we are trying to improve
This was through error-prone PCR

They went back through and looked at the combinations of mutations to see if there was a better mutation to a different amino acid - saturation mutagenesis
After taking a better mutation - they took this type and did an error PCR to find an even better mutation for a larger ‘E’
We end up taking a path but there could have been other/better mutations

However, the route we go through can’t be controlled but it affects what we get at the end - as some mutations wouldn’t have been found if it wasn’t for the path we took

44
Q

How do we measure the preference towards an enantiomer?

A

E (selectivity factor) - if the number is 1 it would favour the 2 enantiomers equally

Drawback - not selecting for the real compound we want
We are selecting an analogue

45
Q

What are some other factors that stereochemistry can affect?

A

Th stereochemical outcome can determine:
By which enantiomer was used
AND
By which reaction mechanism is used

Trying to do directed evolution to change these chemical processes - even harder

46
Q

What can we compare when looking at preference to which enantiomer was used?

A

We can see the different energy levels in two products
Therefore we can get a thermodynamic ratio of the products - we can’t change this
The kinetic ratio - we can alter to change the ‘barrier’ so more of that product is made

47
Q

What does directed evolution with stereoisomers start to overlap with?

A

Computational methods
E.g. Determine a visual landscape of how the reaction moves along

We can see structures that will move along the minimum energy pathway (not up hill)

2. BIOC 2306 S1 (19 decks)

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