Ch E 340 Exam 1 Part 1/2

Front 1

Catalysis

Back 1

A catalyst is a chemical species that decreases the activation barrier of a reaction while not being consumed

Front 2

Inorganic vs organic catalysts (diff rxn)

Back 2

INOR-CAT: isomerization of alkanes, Pt/Zeolite

cond: 15 bar, 250C
-
OR-CAT/ENZYMES:

antibiotic production, polyketide synthetase

cond: 1 bar, 16-37C

Front 3

Inorganic vs organic catalysts (same rxn)

Back 3

Haber process vs Nitrogen fixing

haber: vigorous conditions

nitrogen fixing: in the soil at normal conditions

Front 4

Example of Stabilization of transition states by an enzyme

Back 4

hydrolysis of ATP by myosin

Mg is used to stabilize the negative charge of Pi (Phosphate group)

Front 5

What type of 2 reactions can an enzyme couple?

Back 5

Enzyme can put an exothermic reaction and endothermic reaction right next to each other to drive the endo reaction to happen.

Front 6

What is an enzyme cofactor? what are the 2 main types of cofactor?

Back 6

Cofactors supply enzymes with

energy (work),

e-/H+ (redox),

donor groups (CH3, CO2-, C=O),

Or to solubilize functional groups

There are organic and inorganic cofactors

Front 7

Reaction:

Pyruvate+CoA+(NAD+) -->Acetyl-CoA+NADH+(H+)+CO2

Describe the function of Coenzyme

NAD+ -->NADH

Back 7

NAD+ is the oxidized version

and as it goes through the reaction to

NADH it is reduced, it carries electron to reduce something else later

Front 8

Reaction:

Pyruvate+CoA+(NAD+) -->Acetyl-CoA+NADH+(H+)+CO2

Explain why use Acetyl-CoA

Back 8

Because for an Acetyl group, you can have a good leaving group (Cl-), or bad leaving group (-H)

but CoA 's reactivity is in the middle of the spectrum, therefore we can regulate the reaction more controllably.

Front 9

What is Enzyme Classification (EC)?

Back 9

It is a 4-tier numerical system that classifies the type of enzyme and becomes more specific as the number goes

Front 10

What is EC1,what kind of reaction does it catalyze and its subset

Back 10

Oxidoreductases

(redox reactions)

oxidases, dehydrogenases

Front 11

What is EC2,what kind of reaction does it catalyze and its subset

Back 11

Transferases

(Transfer phosphate, methyl, amino, ... groups)

kinase, mathyltransferase

Front 12

What is EC3,what kind of reaction does it catalyze and its subset

Back 12

Hydrolases

(use water to break bonds)

lipase, amylase

Front 13

What is EC4,what kind of reaction does it catalyze and its subset

What is it opposite of?

Back 13

Lyases

(break C-C, C-N, C-O, C-S bonds ;;; break bonds without water, doesn't move functional groups)

carboxy-lyases=decarboxylase, aldehyde-lyases=aldolase

Front 14

What is EC5,what kind of reaction does it catalyze and its subset

Back 14

Isomerases

(mutates, re-arrange bonds only)

usually have neutral dG thermodynamically

Front 15

What is EC6,what kind of reaction does it catalyze and its subset

what is it opposite of?

Back 15

Ligases

(forms C-C, C-N, C-O, C-S bonds;;; uses ENERGY from eg. ATP)

DNA Ligases, synthetases

Front 16

Advantages of using enzyme as biocatalysts?

rate?

what does it do with types of substrate?

what reacting conditions do they need?

cost?

waste?

can they be altered

Back 16

- rate10^12 fold compared to non-catalyzed

- wide tolerance of structurally diverse substrates

- Regio, Stereo and Enantio selectivity

- Mild reacting conditions

- Waste from bioreactors are easier to clean up

- enzymes can be engineered to increase efficiency

Front 17

what is stereo-selectivity

Back 17

selectively binds to substrates according to their shape

Front 18

what is Regio-selectivity

Back 18

selectively forms / breaks bonds between similar groups (3’ C vs. 5’ C)

Front 19

What is Enantio-selectivity

Back 19

selectively produces an enantiomer (chiral product)

Front 20

Limitations of using Enzyme as catalysts:

are they stable?

can they operate on their own?

are they easily available?

any kinds of investment needed?

Back 20

- Instability of some enzyme in the desired process conditions: but Enzyme engineering can increase stability in new environmental conditions

-Some enzymes require cofactors and cofactor recycling eg. oxidoreductase

- Commercial availability versus cost of DIY

- Investment & Costs in new equipment, training, and techniques:

--If the process already exists, then biocatalysis must be 5-10x better

--If it doesn’t exist, then there is no previous investment

Front 21

Can an enzyme catalyze more than one reaction?

Back 21

Yes they can, lacZ can breakdown anything that looks like lactose.

Front 22

LECTURE 3 LECTURE 3 LECTURE 3

Back 22

LECTURE 3 LECTURE 3 LECTURE 3

- Industrial Organisms

- Review of Macromolecules & Gene Expression

Front 23

Common industrial organisms

Back 23

PROKARYOTES:

E coli, Bacillus subtilis, Lactobacillus, Acidaphilus

EUKARYOTES:

baker's yeast, CHO cells, Algae

Front 24

important structures of Prokaryotes

Back 24

- No internal compartments- Cytoplasm- Cell wall- Peptidoglycan capsule- DNA and RNA concentrated in nucleoid- (gene expression machinery too)- Smaller genomes

Front 25

structures of Eukaryotes

Back 25

Many internal compartments

Front 26

What type of organisms have larger genome than us humans?

Back 26

Plants - they can't move, so have to fight with the environmentProtective chemical defense to things that can attack uswhile human just run away.

Front 27

What are the difference between Animal and Plant cells?

mobility? which compartment rich? what kind of membrane? other characteristics?

Back 27

ANIMAL: motile, mitochondria-rich,thin plasma membrane,many signalling pathways

PLANTS:stationary, chloropast-rich,thick cell wallmany viral & bacterial defenses

Front 28

Types of protein production in Bioreactors

Back 28

1. Mammalian cell culture

2. Photobioreactor for algae

3. Aerobic fermentation - pump O2 in, CO2 out/SPARGING O2 from the bottom to mix

4. Anaerobic fermentation - yeast

5.Light Emitting diodes

Front 29

What are the conditions that will make organisms in bioreactors grow fastest?

What are the usual things bioreactors are for?

Back 29

Have the correct/optimal:

Temperature, pH, dO2, light

Bioreators exist to maintain optimal growing conditions

most of the time need to cool down the reactor bc exo reactions

Front 30

Viruses effect on the bioreactor:

how long does it take to infect 50,000 L Bioreactor modes of viruses

Back 30

only takes 30 minutes per cycle, 6 hours to infect the whole tank (exponential growth)

1. T4 bacteriophage: targets E coli

2. Lysogenic mode: incorporate DNA into hosts, self repression of gene expression til lytic mode

3. Lytic mode: infection, gene expression, enzyme expression, DNA replication of viral DNA

Front 31

Virus structure (bacteriophage)

Back 31

Capsule holds DNA or RNA payload (P=20-40atm)

Hollow delivery Channel

Target recognition domain (binds to receptor on cell's surface)

DNA/RNA ejected into cell with high force

Front 32

color code for each section of a VIRAL CAPSID (shell)

Back 32

blue: protein

green: DNA

red: protein packaging

Front 33

How are DNA packaged into the Capsids

Back 33

Rotating, Pushing and scrunching:

- DNA is threaded through a hole (can bind DNA)

- The proteins grip the DNA nucleotides

- rotated in 12deg increments

Front 34

REVIEW OF BMB REVIEW OF BMB

Back 34

CENTRAL DOGMA

Front 35

What does DNA stand for, what are the parts that make it up and what are the base pairs

Back 35

Phosphate backbone, deoxyribose sugar, nucleotide

ATGC

Front 36

Class about Avastin

Back 36

inhibits ANGIOGENESIS (development of blood vessels)

stop growth of cancer cells that tries to create new blood cells ==> turns of pathway of that disease

Front 37

x-ray diffraction of protein crystal

Back 37

X-ray crystallography is essentially a form of very high resolution microscopy. It enables us to visualize protein structures at the atomic level and enhances our understanding of protein function

Front 38

What are the steps in protein expression

Back 38

DNA --transcription--> mRNA

--translation-->

--Protein folding--> secretion or membrane insertion

Front 39

What are the important RATES in protein expression

Back 39

Rate of Protein production

Rate of Protein folding

Rate of membrane insertion

Rate of Secretion

Production rate of proteins must be balanced with other cellular processes

Front 40

What are the key questions in controlling protein expression

Back 40

1. Which Protein is produced?

2. When is the protein produced?

3. How much protein is produced?

Front 41

Control of Protein Expression:

What controls which protein is produced?

Back 41

AA sequence

Front 42

Genetic parts to controlling protein expression: The Promoter - what is it bounded by? what does it control? what binds nearby it?

Back 42

Controls the rate of transcription

Bounded by RNA poly & Sigma factor (initiation)

Transcription factos can bind nearby to increase or decrease rate

--- Therefore, TC rate can be altered by changing the chemical environment---

Front 43

Genetic parts to controlling protein expression: The Ribosome Binding site - What does it control? what can its result bind to, any special names?

Back 43

Controls the rate of translation (TRL)

- mRNA can be bound by regulatory RNAs to alter TRL

- mRNA can directly bind chemicals to alter TRL too, these are called RIBOSWITCHES

Front 44

Genetic parts to controlling protein expression: The Protein Coding sequence -What does the sequence determine? How many AA's are there? What is the rate of addition? how can it slow down?

Back 44

They determine AA sequence of a protein through CODONS which are series of nucleotide triplets.

- there are 64 codons but of 20 (21) AA's

- ribosome can add 20-30 AA/sec while TRL

- ribosome will slow down if tRNA or AA are not available

Front 45

Genetic parts to controlling protein expression: Transcriptional Terminator - what does RNA Polymerase and mRNA do when it reaches here? what follows after this sequence?

Back 45

This stops transcription

It destabilizes RNA polymerase. This portion of mRNA forms a hairpin that folds quickly, and yanks 5' mRNA out of RNA poly.

- UUUUUUUUU: weakens RNA-DNA hybrid --> dissociate RNA poly

Front 46

Genetic parts to controlling protein expression: Operon - what kind of organisms have this? what does it contain?

Back 46

bacteria can encode multiple ribosome binding sites and protein coding sequences on a single messenger RNA

- 1 promoter,1 mRNA, but expression of multiple proteins

- codes for multiple RBS and CDS

Front 47

How Do I engineer DNA to make more Avastin?

Back 47

Put the protein coding sequence for Avastin into E coli's DNA (becomes a Recombinant organism) and then allow it to grow in a bioreactor.

- MAKE PROMOTER BINDS BETTER

Front 48

If I know the sequence of Avastin what is preventing me from selling Avastin myself?

Back 48

Patent! Intellectual property law

Front 49

What is a Patent

Back 49

legally allowed monopoly, as an incentive to develop things in the first place. You are allowed to obscure the information in the patent.

Front 50

Regulation and genes expression:

Is the level of every enzyme all the same? What are the other 2 proteins? Do we need a lot of them?

Back 50

ENZYME: 10s-10000s for diff type of reactions

REGULATORY PROTEINS: only a few (1-10) needed to regulate the rate of TSC, TRL
STRUCTURAL PROTEINS: needed for the shape e.g. a flagella is a multi-protein complex

Front 51

Gene expression: Range of rates: what is the slowest step in TSC and TRL

Back 51

- for both TSC and TRL the slowest step is the INITIATION step

- Eukaryotes have slower rates

- shorter half-lives in bacteria mRNA and Protein Degradation

Front 52

Gene expression: Rate equations: What is your assumptions? what happens when you add cell growth rate?

Back 52

Assumptions: system reaches Steady State which means d[mRNA]/dt=0 and d[Protein]/dt=0

When cell growth rate is added protein concentration at steady state decreases.

Front 53

Thermodynamics of TSC INITIATION in bacteria.

What binds to the DNA for transcription in bacteria? What are the 4 steps in initiation? How much E does it take to pull apart 2 DNA strands? How is the E barrier overcome?

Back 53

- Sigma factor binds to the DNA for TSC.

1. Initial State

2. Intermediate State

3. Before final state

4. Final state

Gibbs = -35.3kcal/mol (5 times of ATP)

solution: use RNA to ANDCHOR by immediately form RNA-DNA base-pairing with a higher binding energy than DNA-DNA

Front 54

What are the similarities in eukaryote and prokaryote TSC?

What do they use? Where do they start? what is used to code?

Back 54

-both use an RNA polymerase, a multi-protein complex

-both begin transcription (initiation) at the promoter, a recognition-sequence

-the genetic code is the same: DNA to RNA

Front 55

What are the differences in eukaryote and prokaryote TSC?

Initiation complex? rate regulations? gene contents?

Back 55

-Eukaryotic RNA polymerase needs more helper proteins

-Eukaryotic transcription initiation is more regulated (++rate-limiting -steps)

-Eukaryotic genes contain exons and introns (introns removed by splicing)

Front 56

What are the important factors in promoters binding? is the location important? how about the length between?

Back 56

There are strong and weak -10 and -35 Hexamers.

-10 Hexamer has more effect on the rate.

Nucleotide spacer is also important, if too close together won't bind well even if they are strong hexamers.

Front 57

What would happen to protein production if we increase transcription by 10x?

Back 57

Increases by 10-fold.

Front 58

Q: How do we increase the transcription rate using our promoter “toolbox”?

Back 58

We select a promoter sequence whose measured transcription rate is higher.

Front 59

Q: Can we estimate the transcription rate of our current promoter?

Back 59

Sort of, but there is no quantitative model.

Front 60

TRANSLATION what are the highlights?

Back 60

smaller ribosomal unit binds first in both euks and prokes.

tRNA's hairpin loop is the anticodon.

Front 61

Cognate and non-cognate tRNA

Back 61

A cognate amino acid pairs with the tRNA that has the appropriate anticodon; a non-cognate amino acid does not.

Front 62

What is the structure of ribosome 30s when unbound(free) and bound? (30s is the smaller sub-unit) how does mRNA enter the ribosome?

Back 62

UNBOUND: head, body, platform, anti-SD

BOUND: head, body, platform, anti-SD, Ribosomal proteins and bound mRNA. mRNA enters through the "ENTRY" channel

SD= Shine-Dalgarno

Front 63

Engineering the Translation Rate

Q: What would happen to protein production if we increase translation rate by 10x?

Back 63

Increases by 10-fold.

Front 64

Engineering the Translation Rate

Q: How do we modify ribosome binding site sequences to increase the translation rate?

Q: Can we estimate the translation rate of an existing ribosome binding site sequence?

Back 64

- We use a model to design RBS sequences with higher translation rates.

- Yes, using the model.

Front 65

What is the Translation Rate Process? What is the rate-limiting step? what is the equations we can come up with? what do the terms mean?

Back 65

Rate limiting step is the initiation.

Eq:

[mR]/[m][R]=exp(-BGtot)

B=boltzmann

G=Gibbs of ribosome binding

Front 66

Why is Ribosome interactions with mRNA important? How do we calculate the total amount of work needed for R to bind mRNA?

Back 66

Because many ribosome-mRNA interactionscontrol the mRNA’s translation initiation rate.

Calculated by looking at the free energy model/which account for the molecular interactions

Front 67

How does aA Statistical Thermodynamic Model work? What are we finding? What is the equation? Why is it useful? What program do we use for this?

Back 67

We put a whole pool of "Ribosome Ensemble" with R and mRNA, allow competitive binding. We are finsing Rate of translation. it is useful because we can test the model and use it to make quantitative predictions for different applications. We use RBS calculator to find the rate.

Front 68

Why would we have a maxima here?

Back 68

Because cells\ has a finite amount of energy to provide

Front 69

Designing mRNAs:

We want fully folded, active protein.Sometimes, the protein must be secreted.Sometimes, the protein is inserted into a membrane.Q: What could happen if we over-produce a protein?Q: What resources could be depleted?

Back 69

Energy gets wasted