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.