-          There are three of these enzymes that work among four proteins in the body

-          They are unusual because they use the compound tetrahydrobiopterin (a component of folic acid)

o        They provide the source of electrons for the central nervous system to ensure its proper function

-          All work on aromatic amino acids –those that have an aromatic ring as part of their side chain

o        oxygen is attached

-          These three enzymes are: phenylalanine, tyrosine, and tryptophan hydroxylases

** Phenylalanine**

-          You have probably come across phenylalanine before in relation to the disease phenylketonuria, or PKU (newborns are tested for this disease at  birth)

o        Patients with PKU lack phenylalanine hydroxylase, and this makes their phenylalanine become phenylpyruvate instead of tyrosine.  The body cannot metabolize phenylpyruvate because it builds up in the blood and the liver, and eventually it becomes phenylketones, which cause progressive nerve damage. 

o        Once a baby leaves the mother’s womb, the body is no longer able to deal with the excess of phenylpyruvate and will shut down within a few years

o        Treatment consists of diets with low levels of phenylalanine, but is only curable through gene therapy (which has been a little shaky lately due to threats of leukemia L)

o        Consequently there is a real effort right now to find ways to put phenylalanine hydroxylase back into the blood

-          Phenylalanine becomes tyrosine through phenylalanine hydroxylase.  This enzyme introduces a hydroxyl group to the compound.

**Tyrosine**

-          Is a hydroxyl benzyne ring

-          Has been given to patients with Parkinson’s for years

-          Is the first step in formation of dopamine, epinephrine, and nor epinephrine

-          If you don’t have this enzyme, you’re dead

-          There are some people with very mild efficiencies and they can be treated using this enzyme

-          Tyrosine becomes dihydroxyphenylalanine (DOPA) through tyrosine hydroxylase. DOPA becomes dopamine through DOPA decarboxylase.  Dopamine then becomes nor epinephrine through dopamine beta hydroxylase.  Nor epinephrine becomes epinephrine through phenyl ethanolamine s-methyltransferase.  SO, to sum this all up:

§         Phenylalanine à tyrosine à epinephrine (also called adrenaline, produced by the adrenal gland -- epinephrine is our response to stress!)

-          So tyrosine hydroxylase is either turned on or off – if its on it goes on to produce dopamine

-          Here’s a little about dopamine: it is involved in the way we experience PLEASURE

**Tryptophan**

-          Is the first step in formation of serotonin

-          Is found in the brain and also in the stomach

-          There are some mutations in this third enzyme that are responsible for about ten percent of the cases of unipolar depression in this country

-          Tryptophan becomes 5-hydroxytryptophan through tryptophan hydroxylase in a short pathway.  5-hydroxytryptophan becomes serotonin through aromatic amino acid decarboxylase.

-          Here’s a little about serotonin: it effects our MOOD

o        the drug Prozac (for depression) blocks the action of serotonin

o        high levels/low levels of serotonin are related to depression

-          SO the reason we have the enzyme tryptophan hydroxylase is that when we eat food with proteins we break them down into amino acids and then we have to metabolize the amino acids to store them as carbohydrates or fat.  The way we do that with this enzyme is to turn it into tyrosine and then to break it into carbon compounds.

o        This occurs in the liver – is complicated – if you just have a little bit of phenylalanine its not going to do much but if you have a lot it will start to kick in.

-          We know the complete genome of many organisms and can learn a lot about proteins from them

-          We are studying more and more sequences of different genes, and therefore we have a lot of information about the structures of proteins.  Based on those sorts of data you can take the sequences of those genes or proteins and just line them up – you can compare them and get a “really simple picture” in which we break the proteins up into three parts.

-          There is a very distinct similarity (a common ancestor) among the three proteins phenylalanine, tyrosine, and tryptophan hydroxylases

-          For reference, examine the 3D structures of these proteins: the parts known as domains are the structurally distinct parts of the protein.

o        There are three domains – one involved in regulation, two catalytic domains, and four long helices

o        Down in the catalytic domain is where all the chemistry occurs; different amino acids exist in the active site that are involved in the chemistry

o        A lot of the proteins in the cell have metals in them, like iron!  Iron in the active site is good at moving electrons from one place to another.

o        Most of the proteins which have iron in them have it stuck on a large molecule known as “heme.”  Proteins that lack this are known as “non-heme.”

o        We have a greater knowledge about proteins that have heme as opposed to non-heme. 

-          Development of cells is a long term prospect, but there has to be a way to turn this adrenal gland off and on so you don’t produce too much adrenaline at night, for example

-          There are a series of regulators – regulation by phosphorylation

o        somewhere between 1/3 and 1/6 of the proteins in the cell are phosphorylated

o        The way this regulation works is that if you have a cell that makes enough epinephrine, the cell binds and turns it off.  The protein allows this product to come off and it starts the reaction again.

-          How fast dopamine come off the enzyme was measured.  When it was phosphorylated dopamine came off exponentially (very very quickly) – so basically phosphorylation kicks the other stuff out of the way and dopamine comes out of the active site and goes on its way.

-          Scientists propose that the “lid” is closed over the active site so the protein can’t get rid of the dopamine

o        If it is phosphorylated, the lid is opened by a type of kinase

-          The goal is to take proteins and to digest them down into their original amino acids

-          Because we have lots of these, scientists have a lot of material to study – the idea was to target that lid that exists over the active site and to see if they could remove it

o        They used a polyacrilamide gel

-          Basically, phosphorylation occurs faster than your wildest dreams could fathom!!!

-          So all of this is consistent with is the idea that if the enzyme is phosphorylated, it can get into the active site and cut out the dopamine, releasing it quickly J

-          How does one molecule act as glue to keep that door shut?

o        If you look at the ring structure, two oxygens stick on the iron at one end and on the other end the amino group sticks onto the R domain

o        And when you put a phosphate in there you break that part between the amino group and the R group, opening the lid

Just to make sure we’re on the same page with all this:

-          These three proteins (hydroxylases) are similar but are nevertheless very very different in structure

-          You may ask: What keeps phenylalanine hydroxylase from working on tyrosine? What keeps tyrosine hydroxylase from working on phenylalanine?

o        It boils down to evolution of all of this

§         We can go backwards in evolution – identify single gene changes along the way which split the common ancestor gene – we can go back and turn that enzyme that works with “t” into one that works with “p”

-          What Dr. Fitzpatrick did was look at the structures, the active sites of the proteins, again to see what parts of the protein were actually involved

-          Amino acids are identical between phenylalanine and tyrosine

-          There are several compounds in the structure, however, that are different

-          Look at the kinetics of it! 

o        Really simple kinetics scheme – enzyme binds to substrate, then product is eventually formed

o        you can derive the rate at which the product is formed (Vmax – how fast the reaction can carry out its parameters) (Km value – occurs at half the maximum speed) – ratio of a Vmax value to a Km value – velocity is proportional to the substrate concentration is proportional to the velocity over the Km value

-          One single change in the amino acid sequence gives you an enzyme that will produce the other one

IN CONCLUSION

-          What happens is that oxygen reacts with the aromatic ring

-          If in the very end reaction, the hydrogens come together on one singular carbon (on the last step one of them gets lost), they put different isotopes there

o        Hydrogen has three isotopes and they use these and ask in the product, do we have a hydrogen or did it go away to another position?  Either of the two hydrogens can be lost and to know which one has moved, use NMR techniques. 

§         We count the hydrogens – we look for whether we see less hydrogen at a position in which we started with a  hydrogen

ReMeMbEr:  All of this comprises an important system that you need for the functioning of the nervous system.   It’s all quite complicated, to be frank J

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