lectures #1 and #2!! hello. i am tommy wootton. feel free to shut me up any time for questions! bibc...
TRANSCRIPT
Lectures #1 and #2!!
• Hello. I am Tommy Wootton.
• Feel free to shut me up any time for questions!
BIBC 102Metabolic Biochemistry!
Catabolism vs AnabolismTo break down(AKA Cut-abolism!)
To build upAnabolic steroids!
know the 20 amino acids by name, three letter code, one letter code, and be able to recognize (as opposed to
draw) the structures.
Aminoacids:TheMolecularTool Box
fig3-5
(G) (A) (V)(S) (T) (C)
(L) (M) (I) (P) (N) (Q)
(D) (E)
(K) (R) (H)(F) (Y) (W)
Amino Acid/Peptide Bond
Primary Structure - AA sequence
Secondary Structure - 3D structures! ( helix, sheets)
Tertiary Structure - Interactions between these secondary structures
Quaternary Structure - Multiple subunit interactions
Protein Structure
So, lets use a word analogy…
“This guy SUCKS!”
-Hopefully NOT any of you
Primary? --> letters
Secondary? --> words
Tertiary? --> sentence
Quaternary? --> multiple sentences interacting to achieve satisfactory expression of distaste
fig6-2
Energy map of a reaction
G‡ is the activation energy
Activation energy and reaction rate
fig 6-3
4 Main Enzymatic Catalytic Mechanisms
Entropy Reduction
Acid Base Catalysis
Metal Ion Catalysis
Covalent Intermediates
Entropy Reduction
Bringing the pieces together! Forcing them to interact.
Think of Randy’s analogy with 2 cats. Or a cat and a dog.If they’ve got plenty of space, they’re not going to interact. But if they’re brought together…
Acid Base Catalysis
Providing or picking up protons to allow for more efficient reaction catalysis.
Happens in Chymotrypsin! H+ provided by Ser195 OHgroup!
Metal Ion Catalysis
Involves the stabilization of intermediate structuresthrough ionic bonding from metal ion cofactors like Zn2+.
Haven’t talked about it much, but some enzymes do it.
Covalent Intermediates
When a substrate forms a covalent bond with the enzyme to form an intermediate!
Remember TPP (Thiamine Pyrophosphate) or the dihydrolipoyl arm from Pyruvate DehydrogenaseComplex?!
Sample Problem!
freeenergy
reaction
Ligand Binding
The dissociation constant (Kd)
[S][E] [SE]____
S + E (Separate) SE (bound together!)
=Kd
SO…
If Kd is BIG, S and E tend to be separate(HIGH dissociation, get it?)
If Kd is little, S and E will tend to be bound
So back to ligand enzyme binding equation…
If Kd = 0, LB = B (Saturation! Every emzyme is bound)
BUT, if Kd is big (=enzyme and ligand prefer to be separate), LB becomes a smaller proportion of B
What if Kd = L? Hmmm…
Now something cool…
If Kd = L, then LB = 1/2 [B]
So it can tell you (and let you compare!) the affinities of anenzyme to a ligand in different conditions…
Related to the Michaelis-Menton Equation?
Yep! Pretty similar. Except Michaelis-Menton represents enzymatic rates, NOT ligand binding
Vo = Vmax ( ) [S][S] + Km______
LB = B ( ) [L][L] + Kd_____
Same general equation! So, same general shape…
So really, just a change in axes labeling!
- Km = efficiency of RXN - Kd = efficiency of binding
- Gives info about enzyme activity -Gives info about ligand binding
What about a cooperative enzyme?
Due to quaternary structure. Multiple subunits and bindingafinity increases as each is bound.
Makes Vo increase as[S] increases (until you approach Vmaxand saturation.
Ex. Heme vs. Myoglobin
So how fast can an enzyme work?
Vmax = Kcat [E]total
Whats Kcat?
The rate at which an individual enzyme can undergo catalytic function. Essentially it’s how quickeach individual enzyme molecule can do it’s work.
Sample Problem!
M is half saturated when S equals the value of Km… 5 uM
S/(S + Km) = 20/25. So 0.8, or 4/5, or even 80% are acceptable
L is half saturated when S equals the value of Km… 20 uM
The ratio is 1. Since the kcats are the same, the maximal rates (for a given amount of enzyme) are the same.
1. Competitive Inhibitor
2. Uncompetitive Inhibitor
3.“Suicide” Inhibitor
Three Types of Inhibitors!
Competitive Inhibitors
*not quite like this! The dumb@$$ in this picture would be a dysfunctional ligand…
BUT it’s about the concept. They take up space AT the active site.
What does this look like molecularly?
One more view…
Uncompetitive InhibitorsYou’re almost there. At the computer (AKA active site). Ready to go, but someone (inhibitor) is bugging ya.
An uncompetitiveinhibitor binds away from the active site. Doesn’t compete for the active site. Notlike in this pic.
Looks like?
Another view…
“Suicide” InhibitorLiterally takes the computer(enzyme) out of commission.
Covalently binds to the enzyme and breaks it downso it doesn’t work anymore.
:( -Lowers Vmax.-No effect on Kcat
Allosteric regulation
Allo = other, steric = site SO, regulation through binding at a site OTHER than the active site.
Can be inhibitors OR activators. Often the regulator isa product or substrate (allows for negative feedback!)
ChymotrypsinA serine protease! Cuts up proteins! Hydrolyzes its substrates (W, Y, F, L) at the carboxy terminal of the peptide.
W=Tryptophan, Y=Tyrosine, F=Phenylalanine, L=Leucine…
The Catalytic Triad = Asp102, His57, and Ser195.Asp102 stabilizes the positive charge of His57 when itpicks up proton from Ser195 after nucleophilic attack.
The Catalytic Triad = Asp102, His57, and Ser195. Hydroxy group of Ser 195 attacks. Proton transferred to His57. Asp102 stabilizes the positive charge of His57 when it picks up proton from Ser195 after nucleophilic attack.
Lineweaver-Burk Plots!
-Axes are reciprocals
-Tells you Vmax, Km Know where they are!
-Be able to think about it. Talk through it. Shouldn’t just look like random lines!
A couple things to remember:
Competitive Inhibitors!
As [I] increases,Km increases, butVmax stays the same!
This is becauseyou will need moreS to reach 50%saturation (to displaceI), but the max rate can theoretically stillbe reached! Justtakes more S.
Uncompetitive Inhibitors
Here, Vmax decreasesas [I] increases. Also,Km decreases!
This is because I binds to an allosteric site andwhile bound, makesE inactive.
Sample Problem!
1/[S]
1/Vo
-1/Km
1/Vmax
When Michaelis-Menton enzymes are plotted in this manner, the resulting data is a straight line, making it easier to see when an enzyme behaves this way and easier to discern what kind of inhibitor is involved in an experiment.