Basic Concepts in Catalysis

Fundamentals I

August 26, 2008

11:00 – 12:00

Dr. Baggott

· Goes into a spiel about testing big concepts…he relates this to a forest. You will not be tested on the little trees in the forest, only on what’s important and will be on national boards.

· Unless a tree (small detail) is very important, don’t know it. Important things will be pointed out.

· Understand the forest and a couple of trees.

· Healthcare is being more scientific and less-practice oriented.

· For any chemical reaction, there is an equilibrium constant. The equilibrium constant for AàB is

Keq= [B] products/[A] reactants; [ ]= concentration

· Units of concentration: micromoles/liter, millimoles/liter. In biological systems, concentration is not usually over millimoles/liter.

· The keq can be anything; for example

K1= 10 & k-1 = 1 so Keq = [10]/[1] = 10.

· The equilibrium constant (Keq) is actually a function of rate constants…just accept this. It is a ratio of rate constants.

· Don’t need to go through the math….math is in the book.

· CONCEPT: a catalyst put into this system of Aà B does NOT change the Keq…it can’t. The keq is a function of the temperature, the nature of the reaction, and a bunch of other things. The catalyst can’t change the keq.

· If the catalyst can’t change the keq, what does it change? It changes the rate constants. If k1= 10 and k-1= 1 so keq = 10. Add a catalyst and the catalyst will ALWAYS increase both of these proportionately to give you the same keq.

· Example test question: k1 is increased to 1000 by a catalyst, what happens to k-1? The ratio always has to be 10, so k-1 =100 so the keq will stay fixed at 10. The catalyst accelerates the forward and backward reaction such that the keq DOES NOT change. Catalyst allows you to speed up the rate of the reaction.

· Said in a different way, rearranging Keq = [B]/[A] = k1/k-1 = 10 (if we throw in a catalyst the equilibrium constant will stay at 10 regardless…even if k1 or k-1 goes way up or way down…the catalyst accelerates the forward and backward reaction so that the equilibrium constant is not the same

· Catalysts

o Speed up rate of reaction; they do not change the keq.

o Catalysts are not used up; no chemical alternation (just like a sparkplug in a car; Are sparkplugs used up every time they are used to catalyze the combustion of gas in your cylinder? No. They are used over and over again.)

o Catalysts lower the energy of the transition state, from the reactant to the product. This speeds up the reaction.

o Enzymes are proteins; they are biological catalysts; lower the free energy of the transition state; bind substrates and products in the active site. Most enzymes utilize a cofactor or a prosthetic group to perform their activity.

· What is meant by lowering the energy of the transition state? Draws picture on the board.

o No one knows what the x-axis, the progress of the reaction, means. Accept this and move on.

o x-axis – progress of reaction

o y-axis – free energy

o average energy (dashed line) of substrates (higher on graph) and products (lower on graph)

o delta G starts at substrate line and goes to transition state

o adding a catalyst lowers the free energy of the transition state

o In an uncatalyzed reaction, you must go through some sort of transition state.

o The transition state does not resemble the product, does not resemble the reactants, and does not resemble the substrate.

o In biological systems, the reactant is the substrate. They are equivalent.

o All spontaneous reactions that occur from a pure substrate will go downhill (referring to the shape of the graph- from high to low). They go from a higher energy to lower energy. Between the high energy and low energy is the transition state.

o If a catalyst is added, the transition state is lowered; the reaction occurs quickly and more rapidly.

· TEST QUESTION: When an enzyme used acid/base catalysis, what does it do? Lowers free energy of the transition state no matter what mechanism they use.

· KEY CONCEPT: Most enzymes have a molecular weight of 10000 or more. No enzymes are known with a molecular weight less than 10,000. The reason for this is because it can’t create an active site. If you have too small of a polypeptide, you can’t create an active site.

· Most enzymes are spherical. (Draws a circle with an oval shape inside the circle to represent the active site.) The active site is a groove and takes up no more than 10-15% of the surface area. THE ACTIVE SITE IS NOT IN THE CORE OF THE ENZYME, IT’S ON THE SURFACE.

· The enzyme positions catalytic groups, usually side chains of amino acids or cofactors, at the active site.

· A substrate in solution cannot catalyze a reaction because it is not close enough the enzyme’s active site; the substrate must be PHYSICALLY bound to the active site along with its coenzymes and cofactors to work on conversion of reactants to products.

· Lots of enzymes have cofactors; most of them are transition metals, sometimes they are alkali metals like potassium.

· Cofactors = metal ions, mostly transition metal ions.

· Coenzymes

o Small, non-protein molecules that help the enzyme with its reaction.

o Coenzymes are BOUND at the active site to help the enzyme do its work; there is an equilibrium though. Some coenzyme is bound to the active site, some is in free solution. Some substrates are bound and some are in solution.

o Coenzymes that we will be discussing will be biotin, panotothenic acid, riboflavin, NAD, pyridoxine, folate, thiamine.

o Sometimes, the coenzymes have prosthetic groups.

o Prosthetic group- means that there is a covalent bond between the coenzyme and the enzyme. This prevents the coenzyme from diffusing out into the solution.

· Apoenzyme: enzyme without its coenzyme or cofactor bound to it.

· Holoenzyme: Apoenzyme with bound coenzymes or cofactors. When apoenzyme binds its coenzyme or cofactor, it becomes a holoenzyme.

· Vitamin: a precursor to an enzyme; there are a few exceptions, but this is a good general hypothesis.

o Patient asks what’s a vitamin? A vitamin is a precursor and usually there is 1 to many metabolic steps to convert a vitamin into the coenzyme.

o The coenzyme is not ingested by humans; we eat the precursor to the coenzyme (the vitamin). The body does some metabolism to the vitamin and converts it to a coenzyme.

· Reaction types. There are only six types in the body.

1. Oxidation/reduction: a substrate is oxidized and it’s reduced. A substrate that is reduced becomes oxidized as a product. For everything oxidized, there must be something reduced. They are all bisubstrate reactions.

2. Transferase: you transfer a group. One group from one metabolite is transferred to another metabolite. Could be a 1 carbon group, a 2 carbon group, etc.

3. Hydrolases: Just adding or subtracting water from a substrate.

4. Lyases. Removing or creating a double bond. Lyse means remove or create double bonds.

5. Isomerases: optical isomerase (RàS configuration), geometrical isomerases (cisà trans)

6. Ligases: most important, you form covalent bonds. Almost always use energy from ATP.

· Human body does not utilize or do anything with triple bonds.

· Also, in this class, we will only be talking about humans. When we get to metabolism, we will be talking about human or mammalian metabolism.

· All enzymes can be named using their substrates plus an ending of oxidase, reductase, transferase, hydrolase, dehydratase, lyase, isomeras (mutase) or ligase.

Examples:

a. X + ATP à ADP + X – P

b. Name: ATP: X phospho-transferase

c. X(ox) + Y(red) àßX(red) + Y(ox)

· However, we do not use this naming system or the systematic name. Won’t be tested on systematic naming system.

· Three general ways for an enzyme to catalyze a reaction:

· All three of these ways lower the energy of the transition state.

· Known to occur in systems that do not involve enzymes.

· So we know that enzymes utilize mechanisms that are used in organic or inorganic chemistry.

1. Acid-Base: In the active site of an enzyme, it may have side chains of amino acids that can serve as an acid or a base.

a. Acid = proton donor

b. Base= proton acceptor

· Side chain of glutamatic acid, if it’s protinated, it is used as an acid.

· If the carboxyl group is ionized, it will become a base.

· Lysine, Arginine

· Histidine: the protinated form, a portion of which occurs at neutral pH of 7, is a proton donor; the un-protinated is a proton acceptor.

· To a lesser extent, serine and tyrosine.

The big three would be glu, asp (he only said the abbreviations here), lysine and arginine and histadine. If you go into research, you will see many enzymes that use histadine. Also glutamine and aspartic acid, and aspartate – you will see many enzymes bind at their active site to facilitate acid base catalysis.

All acid base catalysis does is lower the free energy of the transition state.

So we have this transition state in the un catalyzed reaction, if we put a proton on it, that lowers the free energy of the transition state. That is what acid base catalysis does. Base would be to take away a proton which would lower the free energy of the transition state.

2. Covalent catalysis – you’ve seen that with chromotrysin for a diagrammatic image of what covalent catalysis is. You have an enzyme that covalently binds its substrate (indicated by 3 dotted lines) during the catalytic sequence; it binds covalently with its substrate, coverts its substrate to product and then you get enzyme plus product.

E + S à E lll S à E lll P à E + P

I told you enzymes are never modified – well they are briefly modified during covalent catalysis, but as it turns out in the whole cycle, they are never modified. You start off with a free enzyme and end up with a free enzyme. (These represent a small portion of the cycle; they are temporarily covalently made.) Modified, but the full reaction sequence allows them to regenerate.

· Figure 9-37 (chymotrypsin), Figure 9-38

Chromotrypsin binds a peptide here, forms a covalently modified acyl-chromotrysin; the acyl group is then released. In the case of chromotrypsin, the covalently modified intermediate here is an acyl coenzyme.

3. Metal ion – usually you have a transition metal at the active site. It kelates the substrate and the products to lower the activation energy of the transition state. It is there to kelate or form weak bonds with the substrate and the products at the active site of the enzyme and facilitates catalysis by lowering free energy.

Good example of that would be carboxyl peptidase A which uses zinc.

· Figure 9-27 (proposed tetrahedral transition state in the peptide-bond hydrolysis by carboxpeptidase A…Zinc).

· Bottom line: table 8-5

Bottom line – add up rate enhancement or how much rate constants can be increased by these various forms of catalysis:

Acid base + covalent+ Metal ion (these are not mutually exclusive; some enzymes use all three of these things) You would get a 106 increase in the rate, or about a million times. That doesn’t even come close to most of these things; not that they are not using all of these mechanisms, but there must be a 4th method.

(Referring to table) 4th mechanism is unique to enzymes; the way we can illustrate the 4th mechanism is by an organic reaction. We will do this because its fun J

Here is a reaction, an organic chemical reaction (refers to 11.12) to form a cyclic ester. This is a benzene ring, this is an acid, and this is a hydroxyl group. When the acid and the hydroxyl group condense to release water, they form an ester. If you look at a space filling molecule – you would see that the carboxyl group is dangling out in space. It has one, two –methylene groups on it – it is dangling out in space. And say this is the hydroxyl group – every once in a while, every once in a while, bang – it hits the carboxyl group and splits off a molecule of water to create the cyclic ester.

The rate constant here is about 10-6. Very slow because it is just wandering around.

Let’s then put some methyl groups on this molecule and methyl groups on one of the methylene linkage to the carboxylic acid. And, let’s induce (put strain on this molecule.) What this does is – the carboxyl group can no longer just rotate around and fling around in space – it is put right next to the hydroxyl group. Right next to it. Right there. Right there where it should be. The rate constant goes from 6x10-6 to 1.5x10-5. The increase is 2x1011. And that is similar to what enzymes do. They force the substrate into a confirmation that resembles a transition state or resembles something that has to react and increases the rate 1012, 1014, 1017 is the most fastest.

Q: What is the number?

A: (Just so we have this on record incase he asks us on the test) Number is not important. Number is not important. You will never be asked a number. That is not even a tree, that is a leaf. The concept is important that the enzyme forces the substrate into a confirmation that resembles the transition state. This won’t be asked on the test; it is just something that organic chemists devised to explain rate enhancement that an enzyme can do.