Lecture 4: Enzymes Flashcards
(21 cards)
How does chemical reaction look like in a graph - without and with an enzyme?
Without = linear trend
- Substrate -> Product
- the more substrate we have the higher velocity
With = hyperbolic graph
- Enzyme+s -> Enzyme-substrate complex -> E + P (irreversible)
- increase -> platau (Vmax) = no velocity gain, maximum velocity (even adding more substrate won’t increase the speed of the reaction)
- no more binding sites, enzymes cannot work harder
- the start resembles the linear trend
Look at the Michaelis-Menten Kinetic. What is Kmax?
- Actual velocity can be calculated with the following formula
- Km = michaelis menten constanr
- upper part explains sum of how fast the enzyme-substrate complex falls apart
- lower part how fast does ES form
-> how tight does the enzyme binds to the substrate (affinity) / substrate concentration at which the velocity is 50% of its max value
NOTE: k2can also be called Kcat = turnover number in moles per second
What does it mean if one enzyme has higher Km than another?
- One enzyme needs more substrate to reach half of its Vmax -> has lower affinity
- Either higher upper part
- faster dissociation from ES (regardless of the direction)S
- Or lower lower part
- rate at which enzyme substrate builds id decreased (lower affinity towards substrates)
Look at how much can enzymes differ in their catalytic abilities. What is meant by ratio kcat/Km?
= measure of the relative efficiencies of the same enzyme acting simultanously on two competing substrates
- i.e. which substrate does it prefer
Why do we use enzymes?
- Allows milder reaction conditions (less pressure, lower temperature, ph)
- Higher substrate specificity
- Possibility of regulation
- binding of molecules (inhibitors, allosteric modulators)
- covalent modulations
- amount of enzyme (transcription, translation)
What does the following tell us:
Look at a specific example of the nomenclature:
How do enzymes bind substrates?
Key-Lock-Model
Substrate and pocket are mostly geometrically and electronically complimentary
- In some cases the enzyme undergoes a conformational change -> and now substrate can bind to the previously noncomplimentary pocket = “induced fit”
- pH optimum
- enzymes often catalyse proton transfer
- most enzymes work the best under physiological conditions (pH = 7)
- High = denatures proteins
Temperature optimum
- higher t speeds up reactions
- BUT too much => denatures proteins
- Depends on organisms
- e.g. some function at low temperature while some at higher (psychrophile, mesophile, thermophile)
What is helping an enzyme? What is Holoenzyme?
- Holoenzyme = form where the enzyme is ready to bind substrates
- Apoenzymes = the enzyme itself
- Cofactor = helpers
- Metal
- Coenzyme = organic
- covalent affinity = prosthetic group
- easy to dissociate = cosubstrate
Coenzyme - e.g. ATP (made by the body), essential vitamins (taken up from food)
Again - how does step-by-step procedure from enzyme to product work?
How do enzymes help from the thermodynamics stand of point?
Enzymes accelarate chemical reactions by lowering activation energy (investing energy) needed for the transition (it lowers the transition state)
- The further down the P is the more we get out
Look at examples of enzymes in transition states:
What kind of chemical reaction do we get by adding Serine Proteinases? What is substrate and product? What family does it belong to?
- Reaction starts with polypeptide chain which gets cut into 2 peptides by the means of hydrolysis
- Belongs to chymotrypsin superfamily e.g. chymotrypsin, trypsine, thrombine
- Peptide bond or Scissine bond
Explain the reaction mechanism of Serine Proteinases
Serine proteinases forms an enzyme pocket with serine that reacts to the substrate and base of histamine
-> serine binds to the substrate -> histamine can be under these conditions protonated i.e. H+ gets added -> which allows for the creation of Tetrahedral transition state
-> H+ of the histamine gets attached to the N-terminus and forms the first peptide = Acyl-enzyme intermediate
-> H2O attacks the remaining complex of enzyme-substrate -> donates H+ to the base and -OH to the substrate -> severes the bond with serine -> second peptide
-> enzyme returns to normal position (negative charge of serine + positive H+ get closer)
What makes histadine more likely to be protonated (=> Catalytic triade)?
There is actually an additional Asperate with negative charge which collaborates with Histadine to pull H+
-> Catalytic triade = Asp, His, Ser
What else is needed for the reaction to occur?
- We also need Oxyanion hole = structure in close proximity to the catalytic triade that stabilizes negative charge in order to push the reaction forward
- Main chain interactions
- Specificity pocket (limit number of molecules that could be considered the enzyme’s substrates)
Look at structure of Chymotrypsine.
Consists of 2 domains - anti-parallel beta strands, 6 beta strands per domain
- precurser = chymotrypsinogen created by cleaving of specific chains
- stabilized two disulfide bridges
Look at the active side typology of Chymotrypsine.
- Domains
- Four of the beta strands form Greek Key and antiparallel hairpin motif
- Active site is at the intersection of domaons -> ser, hist, asp
Can we inhibit chymotrypsine?
- Non-covalent inhibitor
- part of the peptide gets cleaved -> however the rest makes quite strong bonds to the enzyme which will keep holding it in place -> ultimatelly inhibiting the active site
What does the specificity pocket do again?
= enable active sites to accept only specific molecules
- picture depicts different serine proteinases -> each has different preferences e.g. trypsin = negative so it will attract positive molecules
