Enzymes Flashcards
What are enzymes (overview)
- proteins
- catalyze the chemical transformation of substrate (S) to a product (P)
- increase the rate of reaction
function by lowering the activation energy of the reaction
E + S <–> ES <–> EP <–> E + P
- two intermeidates
What is the michaelis- menten equation
V0 = ( Vmax x [S] ) / (Km + [S] )
- there is a max speed that chem rxn can occur, notice how simialr it looks to hemoglobin oxygen binding curve
as you inc concentration of substrate RXN goes faster
What are kinases
X-OH + ATP <—> X-OPO32- + ADP + H+
- an enzyme that caalyses the transer of phosphate groups from higih energy phosphate donating moleucles to specific substances
- this procuess is known as phosphorylation
substrate gains a phosphate group and high energy ATP molecule donate phosphate group
*addition of phosphate groups makes the molecule target for another enzyme
what is Phosphatase
X-OPO32- + H2O <—-> X-OH + HOPO32-
- enzyme that uses water to cleave,a phosphoric acid monoester into phosphate ion and alcohol
bc phosphatase enzyme catalyzes the hydrolysis of its substrate its a subcategory of hydrolases
*kinase is energy dependent but phosphatease gets it from water
what is phosphorylase
X-O-Y + HOPO32- <—> X-POP32- + Y-OH
- enzymes that catayse the addiiton of phsophate group from an inorganic phosphate (phosphate + hydrogen) to an acceptor
*outcoe basically same as kinase
What is Hydrolase
X-O-Y + H2O <—-> X-OH + Y-OH
commonly perform as biochemical catalyst that uses water to break a chemical bond
typically dividdes a larger molecule into two smaller molecules
what are regualtory enzymes
- Multi-step metabolic pathways, contain at least one rate-limiting ste
- catalysis of rate limiting steps is mediated by regulatory enzymes (these enzymes are regulated)
- cataltic rate of these enzymes is controlled by specific signals
*adding another level of control over chemcial proces in a cell by regualteing the enzymes that regulate the reaction
explain feedback inhibitation
Inhibition occurs at the first step of the pathway
No other products cause inhibition
Isoleucine does not bind to the active site
Instead there is a separate allosteric binding site
Inhibition is reversible
*negative feedback, the product inhibits the enzyme that generates it
*inhibitor deosnt bind to active site it binds to a seperate allosteric binding site
what are the mechanisms of enzyme regulation
- allostery
- reversible, non covalent binding of regulatory compounds
- allosteric modulators
- Reversible colanet modifcation
- mediated by a separate enzyme system
- interaction with regulatory proteins
- proteolytic cleavage;
- non reversible, chewing enzyme up and degrading it or putting into diff subunits
What is an allosteric enzyme
A regulatory enzyme with catalytic activity modulated by the noncovalent binding of a specific compound at a site other than the active site
* undergo confomational changes in response to modulator binding, think this is like when one oxygen molecule binds changing from T to R state and changed adjacent subunits
Properties:
- larger, structurally more complex
- usually multi subunit
- have regulatory (allosteric) sites
- bind modulatory
- undergo conformational changes in response to modulator binding
- do NOT obey michaelis- mentin kinetics
what does binding of allosteric modulator do
(how does it change a graph)
- do NOT obey michaelis-mentin, can change K0.5 OR Vmax
- get sigmoidal cruve of V0 vs [S]
- influenced by cooperative substrate binding and /or binding of allosterid modulator
- do not have Km use K0.5
Km = half saturation constant
K0.5 = [S] resulting in 1/2 Vmax
Positive modulator: switch the T to R state
Negative modulator: might switch R state to T state
*sigmoidal bc combination of T and R state
- graph shows spepd at which reaction is going not occupancy, X axis is the same but Y axis different
what the the types of modulators
homotropic: both modulators and substrates, when expose enzyme to substrate you inc speed and affinity of reaction
heterotropic: modulatoes are not substracts, other bidning factors that dictates how the enzyme is able to execute the chemical reaction
what are the effects of modualtors
can have negative effects (reduce activity) or positive (inc activity)
-can affect K0.5 or Vmax
both homo and heterotropic modulators have one of these effects
what is ATCase
aspartate transcarbamoylase: ALLOSTERIC ENZYME
commited step in biosynthesis of pyrimidine nucleotides (ex CTP)
- form N-carbamoylaspartate and Pi from carbamoyl phosphate and aspartate
*has both T and R state (inactive T and active R)
- c nucleotides that this enzyme makes, CTP (ALLOSTERIC MODULATOR)
what is the aspartate transcarboamoylase reaction
- ATCase transfers an activated carbamoyl group onto the amine group of aspartate
this is the first step for synthesizing puring rings (ex CTP)
what is the subunit structure of aspartate transcarbamoylase
- ATCase has two distinct types of subunit: catalytic and regulatory
- the enzyme contains 12 total polypeptide chains: 6 regulatory chains and 6 catalytic c chains
*subunits are individual peptides,
- 3 c chains form each catalytic trimer (c3)
*we have 2 catalytic trimers bc 6 c chains total, 2 catalytic sites
- 2 r chains forming regulatory dimer (r2)
*3 sites of regulation
- subunits interact via zinc domains of regulaotry subunit: zinc domains have cysteines coordinating a structural zinc ion
how was the subunit structure of ATCase determined
via treatment with a cysteine modifying mercury compound and ultracentrifugation
- run thorugh a size separating devise, if put on a lot of mercury to dissociate it we see two peaks, r2 and C3
describe the allosteric modulation of ATCase
- substrate binding by ATCase is explained by allosteric cooperativity T -> R state transition
- aspartate is a positive homotropic modulator
What does CTP and ATP do in terms of being modulators
ATP = +ve heterotopic
CTP = -ve heterotropic
- CTP functions in feedback inhibition
- ATP and CTP binds to sits other than the active site (on the regulatory subunit)
*CTP is a negative modulator, binds to regualtory site and reduces the activity
*ATP is an activator and does opposite
how are substrates and heterotropic modulatoes bound by ATcase different
*structurally very different
- substrates bind at interfaces of c chains (center of complex)
- modulators (ATP, CTP) bind to r chains (periphery of complex)
explain the allosteric efects of ATCase
arise from changes in quaternary structure
- Tstate: ATCase has a more closed conformation
- this closed conformation blocks active site loop from adopting a fully active conformation
*key word is fully active, has some potential for activity
- R state: ATCase is in a more open conformation
- allows active site loop to adopt an active conformation
Howdo we push ATCase into the R stae so we can characterize its structure and properties
- add substrates (only gives short window to study in active state becuase substrate will be used up)
- add substrate analogue that mimics both substrates PALA (mimics substrate and is non reactive, pushes ATCase into R state and keeps it there)
add activator ATP
what are the changes in strucutre of ATCase
what is PALA
N-(Phosphonacetyl)-L-aspartate
nonreactive bisubstrate analog that mimics the reaction intermediate of ATCase
- binds in catalytic site but chemical reaction does not contiue to final product and does not get released
- shoves ATCase from t to r state
what is the strucutal change of ATCase when PALA binds
*dramatic changes in quaternanry strucuture
what are the strucutral changes during activation fo ATCase
- within catalyic subunits
- flexing at interfaces between c chains
- Overall
- catalytic trimers move apart 12 Å and rotate relative to each other
- regulatory dimers roate
- change in bend between r chains`
what happens when CTP bidns to ATCase
- stabilizes T state
(substratte)
what happens when you throw in activator in with ATCase
T -> R appears to be concerted
ATP binding can induce T -> R conversion in absence of substrate
*this change may not be permanent
how can enzymes be regulated by reversible covalent modification
Enzyme activity can be altered by the covalent addition of modifying group(s)
Modification is reversible
Covalent modifications and their removal are mediated by a separate enzyme system
what are the target residues for adenylation
Adenylylation = (Tyr)
What is the target residue for phosphoryation
Phosphorylation = (Ser, Thr, Tyr, His)
what is the target residue for ADP-ribosylation
ADP-ribosylation = (Arg, Gln, Cys, diphthamide)
WHat is the target residue for ubiquitation
Ubiquitination = (Lys)
what is the target residue for methylation
Methylation = (Glu)
what is the target residue for acetylation
Acetylation = (Lys, amino-terminus)
how does phosphorylation occur?
* ATP is the source of the phosphate
how are enzymes regulated by reversible phosphorylation
- Amino acids modified: Ser, Thr, Tyr, His
- Attachment of the phosphoryl group is catalyzed by a protein kinase
- Phosphorylation is sequence-specific
- Phosphoryl group can be removed by phosphoprotein phosphatase
what are the site of protein kinase A
Protein kinase A (PKA) phosphorylates proteins at sites containing the sequence:
- X-R-[RK]-X-[ST]-B
- e.g. F-R-R-L-S-I
Substrates of PKA contain an amino acid sequence that fits this pattern:
- [RK] = Arg or Lys is acceptable;
- [ST] = Ser or Thr is acceptable (and is the residue phosphorylated) X = any amino acid
- B = any hydrophobic amino acid,
X-R-[RK]-X-[ST]-B will be recognized by the catalytic site of PKA
This sequence will then be phosphorylated on the serine or threonine residue
*PKA regualtes by phophorylates its target,
how is PKA regulated
- by regulatory subunit binding
- The catalytic subunit of PKA (c; blue) is generally bound by a regulatory subunit (r; red)
- This regulatory subunit blocks the catalytic active site, inactivating PKA
- Regulatory subunits of PKA contain the sequence K-R-R-G-A-I
This sequence binds the catalytic subunit because it (almost) matches the pattern X-R-[RK]-X-[ST]-B… - But it cannot be phosphorylated (Ala instead of Ser/Thr), Ser/Thr does nothitng to actually attract protein kinase A but is just wahts phosphorylated
how is PKA activated
- by cAMP
- Many cellular signaling events will trigger cAMP production
- The regulatory subunit will bind two cAMP molecules
- The regulatory subunit then releases the catalytic subunit, allowing it to phosphorylate substrates
- AKAP= A kinase anchoring protein, bidns to palsma membrane of cell, it binds regulatory subunits of PKA
*PKA in inactive form sits on plasma membrane, reacts to extracellular signalling
*once activated itll phosphorylate any targets
what does PKA do?
comment on the lifetime of cAMP
- short lived bc hydrolyzed by cyclic nucleotide phosphodiesterase
what do phosphoryl groups introduce
1) A relatively bulky group (can result in steric exclusion)
2) Charge – allowing electrostatic interactions (attraction, repulsion)
3) Oxygen atoms that can participate in hydrogen bonds
4) Site for protein-protein interactions
