Enzymes and Kinetics Flashcards
What are enzymes?
proteins that increase rate of reaction (accelerate transformation of substrate into product). Most are globular proteins.
Strict specificity by weak interactions by the ES
they are characterized according to reactions they catalyze
“ase”
don’t alter chemistry of reactions
What is a cofactor?
non protein compound that is bound to a protein and required for the protein’s activity. It completes the active site of the enzyme by binding to one or more of the amino acid side chains)
metal ions from minerals in food have various functions in catalysis
ex: Fe and Cu are used by oxidases in REDOX reactions
“helper molecules”
what are coenzymes?
Acts as carriers of functional groups.
one of the types of organic cofactors (loosely-bound cofactors)
vitamins
What are the 6 enzyme classifications?
- Oxidoreductase
- Transferases
- Hydrolases
- Lyases
- Isomerases
- Ligases
What are oxidoreductase?
catalyzes REDOX (transfer of e-)
ex: dehydrogenase, oxidase, reductase, catalase
What are transferases
catalyzes transfers of groups (ie. methyl, acetyl, phosphate)
ex: methylase, polymerase, protein kinase
What are hydrolases?
catalyzes hydrolysis reactions where a molecule is split into two or more smaller molecules by adding h20
What are histone acetyl transferases?
enzymes that acetylate conserved lysine amino acids on histone proteins by transfering an acetyl group from acteyl CoA to form E-N-acetyl lysine
linked to transcriptional activation
What are lyases?
- enzymes that catalyzes the addition of groups across a double bond
OR - elimination of groups to generate DB
What is Enoyl-CoA hydratase
enzyme that hydrates the DB between the second and third carbon on acyl-CoA
essential to metabolizing fatty acids to produce acetyl CoA and energy
What do isomerases do?
catalyze atomic rearragements within a molecule.
Ex: Prolyl isomerase is an enzyme that interconverts cis and trans isomers of peptide bonds
What do Ligases do?
catalyze the rxn which joins two molecules
uses energy derived from ATP
ex: peptide synthase, DNA ligase
What are the 2 ways a cofactor can bind to the apoenzyme?
- via a non-covalent bond. Can dissociate from the apoenzyme
- interact via covalent bond. Can’t dissociate from enzyme even after denaturation
what are prosthetic groups?
tightly bound cofactors
Difference between apoenzyme and holoenzyme? Which has low or high enzyme activity?
apoenzyme: inactive enzyme without the cofactor. Has low activity
holoenzyme: complete enzyme with cofactor. Has max activity
What are the 2 classes of cofactors?
- inorganic: metal ions (ie. Na+, Fe2+)
2. organic: coenzymes or prosthetic groups. Also referred to as vitamins
Use of vitamin C (ascorbic acid)?
vital for collagen synthesis
cofactor for prolyl hydrolase and lysyl hydroxylase
Use of folic acid?
cell growth and multiplication
required for nucleic acid synthesis
deficiency results in anemia
sources: leafy greens, liver, yeast, wheat germ
Use of Thiamine (B2)?
found on membranes of neurons
muscle disease (heart)
used in the breakdown of energy molecules
deficiency causes beriberi
Use of vit K?
needed for prothrombin formation, posttranslational modification of proteins, blood coagulation
deficiency leads to increased clotting time
deficiencies are rare
how do enzymes accelerate reactions?
they stabilize the unstable transition state intermediate of the rxn.
Reactants are positioned in the reactive conformation in the active site which facilitates the bond making/breaking when substrate becomes product
what is the active site?
pocket on enzyme where rxn occurs
what is the substrate?
molecule bound to active site. It is acted upon by the enzyme
What is the energy of activation?
amount of energy needed to acheive the transition state.
Requires heat input
which is higher? enzyme activation energy or chemical activation energy?
chemical activation energy is higher. (because enzymes lower the amount of energy needed)
what does -delta G mean?
rxn is favourable and spontaneous.
This does tell how fast the rxn goes
What does every of activation tell you?
high EA = slow rxn
low EA = fast rxn (accelerates faster)
what does +delta G mean?
rxn is not favourable and not spontaneous
how do you accelerate a rxn?
increase thermal energy/ temp or pressure (this increases movement which increases # of collisions)
what are the 5 proteases?
chymotrypsin trypsin elastase thrombin substilisin
What is the serine protease family?
consists of aspartic acid (asp102), histidine (his57) and serine (ser195) residues
participate directly in the cleavage of the peptide bond
“charge transfer relay network”
What is the amino acid preference of cymotrypsin?
Phe, Trp, Ile, Try, bulky sidechain
What does delta G = 0 mean?
equilibrium (no net change)
what is the rate limiting step
highest energy point in rxn.
determines the rxn rate
varies with rxn conditions
what are catalytic pockets on the active site?
they are lined with amino acid residues that bind to the substrate
Wht is the amino acid preference of trypsin?
lys
arg
long sidechain
positive charge
amino acid preference of elastase?
ala
gly
val
small side chain
what is a scissile bond in polypeptides?
a covalent chemical bond that can be broken by an enzyme
what happens if you change one amino acid in the catalytic triad mechanism?
the negative charge will not move down
the length of the side chains would change which changes the specificity pockets
what is the importance of the shape of binding pockets?
the shape of binding pockets determines the types of substrate that can bind and be cleaved
how many aceyl intermediates are formed in the serine proteases?
2
where does chymotrypsin cleave?
cleaves on the carboxy terminal of the amino acid.
Preference for phenylalain, tryptophan, tyrosine, isoleucine
where does trypsin cleave?
cleaves specifically on carboxy terminal of lysine and arginine ONLY
where does elastin cleave?
cleaves on carboxy terminal of small side chain amino acid (glycine,alanine,valine)
what are the 3 factors that affect rxn rates?
temp
ph
enzyme concentration
How does temperature affect rxn rate?
increasing temp = increase rxn rate
q10 rule
What is the Q10 rule?
enzyme activity doubles with every 10deg C increase until approx 40deg C when proteins start to denature
what is the temperature for max activity of most enzymes?
25-37deg C
define reaction rate
the measure of how fast a product is generated
can look at how fast the substrate is depleted
What is the effect of pH changes on rxn rate?
extreme ph denatures proteins (the pH optimum reflects the enzymes biological environment)
amino acid required for catalysis are ionized
substrates become ionized and no longer bind to the enzyme
how doe enzyme concentration affect rxn rate?
rxn rate increase linearly with amount of enzyme (as long as the enzyme concentration is much lower than the substrate concentration)
what conditions do you need for molecules to react with each other?
need to have correct orientation and enough energy to crash into one another
rate = ?
rate = k [reactants] = Kf [A] [B] = V0 = totalproductformed/time
what is k
rate constant
what were michaelis and menten’s observations?
- at constant substrate conc and variable enzyme conc: rxn velocity increases with the enzyme concentration
- at constant enzyme concentration and variable substrate concentration: non linear hyperbolic relationship
what is the michaelis-menton equation?
v0 = (vmax [S]) / (Km + [S])
what are the two parts of enzymatic rxns?
binding (formation of ES complex) and catalysis (breakdown of ES to products)
what are the 2 meanings of Km (michaelis-menton constant)
- substrate concentration at which 1/2 the active sites are filled
[S]»_space; KM when v0=vmax
[S] = KM when v0=1/2 vmax
- dissociation constant for ES when k2»K3
km = k2/k1 = k3
what is the km equation in normal circumstances?
km=(k3+k2)/K1
what does a lower km mean?
lower km means tighter substrate binding
km tells affinity between enzyme and substrate
what is the km range? (what is considered weak or tight binding?)
10^-2 is weak
10^-7 is tight
when is vmax reached?
when [s] is very high
[E] is very low (enzyme is saturated with substrate)
all enzyme is in ES complex
what is v0 when [s]=km?
v0=1/2vmax
what is v0 when [s]»_space; km?
v0=vmax
what is the rate determining step?
slowest step in the rxn
determines the rate of the overall rxn
overall rate of rxn is proportional to the concentration of [ES]
ES E+P (product formation)
what does low [S] mean?
most enzyme is uncombined (high [E]).
As [S] increases, equilibrium is pushed towards ES formation
what happens as [s] increases?
equilibrium is pushed towards ES formation
what is the presteady state?
when the enzyme is first mixed with substrate and [ES] builds up
E+S –> ES
this is a very short period
what is the steady state?
when [ES] and [intermediate] remains constant
this is when [ES] is formed and broken down at an equivalent rate
equilibrium: E+S ES
what does Vmax describe?
- the efficiency of catalysis
ES –> E+P
vmax = k3 [E]
- turnover rate
Vmax/[E] = K3 = Kcat
what does Km describe?
how tight the enzyme binds to substrate
affinity between enzyme and substrate
when is the steady state assumption valid?
when [s]» [E]
what does a higher km mean?
high km means looser substrate binding
what is the turnover rate?
of substrate molecules converted to product when [E]«
how do you determine km and vmax experimentally?
- have a fixed amount of enzyme
- add into test tubes in increasing substrate conc
- incubate
- measure product formed (gives the initial velocity
- plot velocity vs substrate
what is the relationship between v0 and [s]?
directly porportional
what are the axis’ in the lineweaver-burk graph?
1/v0 vs 1/[s]
what is the linewear-burk equation?
1/vo = (Km/Vmax) (1/[s]) + (1/Vmax)
what is the x intercept on the lineweaver-burk graph?
x int = intercept on 1/[s] axis = -/Km
what is the y-intercept on the lineweaver-burk graph?
y int = intercept on 1/Vo axis = 1/Vmax
what is the slope on the lineweaver-burk graph?
slope = km/vmax
what are the four types of enzyme inhibition?
competitive
noncompetitive
uncompetitive
irreversible inhibition
what are the types of reversible inhibition?
competitive noncompetitive uncompetitive
whats the difference between reversible and irreversible enzyme inhibition
reversible: inhibitor doesnt covalently bond to enzyme
irreversible: inhibitor covalently binds to enzyme
what is enzyme inhibition
interference with the catalysis
describe competitive inhibition?
inhibitor competes with substrate for the enzyme’s active site.
the inhibitor is structurally similar to the substrate and occupies the active site. This prevents the binding of substrate to the enzyme
EI complex is formed. No catalysis occurs
In competitive inhibition, does KM and Vmax increase, decrease or stay the same?
Km increases
Vmax is the same
In noncompetitive inhibition, does KM and Vmax increase, decrease or stay the same?
Km is the same
Vmax decreases
In uncompetitive inhibition, does KM and Vmax increase, decrease or stay the same?
Km and Vmax decreases
What is non competitive inhibition?
the inhibitor binds at a different site from the substrate active site (binds to E or ES)
K3 doesn’t occur
What is uncompetitive inhibition?
inhibitor binds at a site distinc from substrate active site (binds only to ES)
doesn’t bind to E
works best when [S] is high
What is irreversible inhibition?
inhibitor binds covalently or noncovalently to the enzyme.
The enzyme is permanently inactived (ie. confirmation is changed)
E + I –> EI (can’t go reverse direction)
when does uncompetitive inhibition work best?
when [s] is high
