Scholz: Enzymes Flashcards
What is a co-factor?
Non-protein component needed for activity (eg: metal ions)
What is a coenzyme?
What is a prosthetic group?
Covalently bound (or tightly associated) co-factor - for example in the Haem group
What is an apoenzyme? What is a holoenzyme?
What is the name of the molecule acted upon by the enzyme? What part of the enzyme does this molecule bind to?
Apo = protein component of an enzyme that contains a co-factor
Holo = whole enzyme (inc co-factor)
Nearly all enzymes end with the suffix ____
-ase
International Union of Biochemistry and Molecular Biology (IUBMB): six classes of enzymes. What do they do?
1) Oxidoreductases ____
2) Transferases ____
3) Hydrolases ____
4) Lyases _____
5) Isomerases ______
6) Ligases _____
1) transfer electrons
2) transfer groups
3) perform hydrolysis (transfer groups -> water)
4) Form (or add groups to) double bonds
5) Transfer groups with molecules (form isomers - molecule w/same atoms)
6) Formation of C-C, C-S, C-O, and C-N bonds (coupled to ATP cleavage, often also forms water)
International Union of Biochemistry and Molecular Biology (IUBMB): six classes of enzymes. What are the names of the enzyme classes that…
1) transfer electrons
2) transfer groups
3) perform hydrolysis (transfer groups -> water)
4) Form (or add groups to) double bonds
5) Transfer groups with molecules (form isomers - molecule w/same atoms)
6) Formation of C-C, C-S, C-O, and C-N bonds (coupled to ATP cleavage, often also forms water)
1) Oxidoreductases ____
2) Transferases ____
3) Hydrolases ____
4) Lyases _____
5) Isomerases ______
6) Ligases _____
Enzymes DO what three things?
What don’t they do? [2]
DO: increase spontaneous reaction rate, lower activation energy, accelerate movement to equilibrium
DO NOT: move equilibrium, make non-spontaneous reactions spontaneous
Spontaneous reactions must have a _______ G value as they will _________ enthalpy (H) and/or _______ entropy (S).
What is the formula for delta-G?
Negative, decrease, increase
delta-G = delta-H - delta-S*T (if temperature constant)
Activation energy represents the energy of the ___________ state. What does this represent?
Transition (energy required to position chemical bonds correctly, bond rearrangements, electron rearrangements, etc) - the moment that chemical bonds are formed and broken.
Enzymes form __-______ bonds with _________ molecules, called the “______ _____”, allowing them to take the reaction through a different path of reaction intermediates.
non-covalent, substrate, binding energy
How do enzymes reduce activation energy? [3]
Entropy reduction (enzymes force substrate(s) to be correctly orientated and keeps them in small space, no longer whizzing around)
Desolvation: weak bonds between substrate and enzyme replace most or all of H-bonds between substrate and aqueous solution
Induced fit: conformational changes occur in protein structure when substrate binds
The enzyme’s structure needs to fit the _______ ______ for it to work properly
transition state
How do you analyze enzymes? [3]
Enzyme kinetics (Vmax, Km), 3D structurers (eg: x-ray diffraction), mutagenesis (mutate gene that creates enzyme, see what happens)
What is V0? What happens to this if you increase substrate concentration?
Initial velocity (it increases)
When you vary substrate amount, get numerous initial velocities, what can you then graph?
What is Vmax?
What is Km?
Michaelis-Menten kinetic
Vmax = the point at which [S] is so large that V0 changes are vanishingly small/zero (enzymes all full up with substrate)
Km = substrate concentration at which you have 1/2 Vmax
At low [S] (below Km), you get an almost ______ increase in V0 as [S] ______
At higher [S], V0 changes ______ in response to [S] increasing
When [S] becomes so large that changes are negligible/zero, you hit _____
linear, increases
little
Vmax
What is the maximum velocity of an enzyme called?
What is the point at which half of this velocity is reached? What does this represent?
Vmax
Km (stability of enzyme-substrate complex)
E + S <-> ES -> E + P
This is the basis of the ______-______ equation
It states that the first part of the reaction occurs _______, and that the second part occurs ____ ______ than the first part
The rate limiting step is the _________ part, so the overall rate of reaction must be proportional to the amount of ______
Initially, there is a period where ____ is at a steady state. Why is this important?
Michaelis-Menten
reversibly, more slowly
second, ES
ES (it remains at the same amount: as ES is converted to E+P, more ES is made = V0)
What is the Michaelis-Menten equation?
What do the elements of the equation stand for?
V0 = Vmax[S}/Km+[S]
V0 = initial reaction velocity
Vmax = maximum reaction velocity
[S] = substrate concentration
Michaelis Menten Equation (V0 = Vmax[S]/Km + [S])
At low [S], you can…
At high [S], you can
Low [S} = ignore [S] in the denominator
High = ignore [S] and Km throughout
Michaelis-Mention
Km is equivalent to what?
The substrate concentration at which the initial reaction rate is half of the maximum reaction rate
Michaelis-Menten
It’s difficult to ever actually figure out Vmax manually. Why? What plot gets around these issues?
Can you remember the the equation? How do you calculate Vmax and Km from the graph (see overleaf)?
Because the graph continues to infinity, and it is difficult to ever experimentally achieve the true Vmax. Answer: Lineweaver-Burk (double reciprocal) plot)
1/V = (Km/Vmax)(1/[S]) + 1/Vmax
Vmax = 1/[1/V] [at intersect of y], Km = -1/[1/S]
Looking at these values, what is the stability of the enzyme-substrate complex (the “fit”) and how fast (enzyme catalytic rate) does it convert the substrate to product (or, rather, from ES to E + P)?
CA: very poor fit, very fast enzyme
CT: poor fit, slow enzyme
PC: good fit, fast enzyme
LZ: very tight fit, very slow enzyme
Glucokinase and hexokinase: what are they? How do their Km and Vmax differ? Why is this important?
Both catalyze Glu + ATP -> G6P + ADP (Glu in liver, hex elsewhere) [G6P is “trapped” in the cell]
Glu = High Km, high Vmax, Hex = low km, low vmax
Blood glucose up -> glucokinase activity goes up slowly, whereas hexokinase is already maxed out (liver should only capture glucose when it is high, whereas rest of body will be wanting to keep it in the cell all the time). Blood glucose low -> glucose released from liver, but glucokinase unable to phosphorylate it, so it can go into the blood and into cells, where hexokinase is able to “trap it”
Enzyme with two or more substrates… how can this occur? [2/1]
Lactate dehydrogenase exhibits an ordered sequential mechanism to its catalysis of:
pyruvate + NADH + H+ <—> Lactate + NAD+
So how does this play out?
Enzymes with 2+ substrates: amino groups shuttling between amino acids and ketoacids are examples of reactions that have no ternary complex. What is Cleland notation, and what would it look like in regards to the reaction of aspartate + alpha-ketoglutarate to oxaloacetate + glutamate?
Extra points: what is the name for this kind of reaction (enzyme: aspartate aminotransferase)?
Cleland notation is what you use to demonstrate binding order in reactions
(double displacement/ping-pong)
Competitive inhibitors of enzymes exhibit _______ Km. What happens to Vmax? Why?
Where would the line move on Lineweaver-Burk plot?
Increased Km (weaker enzyme-substrate complex/less tight fit). Vmax stays the same (because you can increase substrate concentration to overcome the inhibition).
It would tilt to the left (passes through Y at same point, passes through X closer to zero)
What do non-competitive inhibitors do to Km and Vmax?
What will the lineweaver burk plot look like?
Substrate still able to bind, so Km remains unchanged. However, increasing substrate concentration does not change inhibition, so Vmax will decrease.
Line pivots to the left (around the X axis)
What weird kinetics do allosteric enzymes show?
What two models are there to explain this weird curve?
Flattened S-shape curve : increases in [S] at first give only small increase in V0, then much larger ones, and then more slowly again.
Concerted model & sequential model
Why might having multiple phosphorylation sites be useful?
Allows fine control of enzyme function (dependent on how many sites are phosphorylated)
