The reaction is basically shown below. Dopamine hydroxylase is an enzyme, so I'm not sure if we can have a theory based on organic chemistry. On the other hand, maybe there is a theory that involves structural biology?

Evolutionarily speaking - the enzyme has evolved to add OH to the beta-carbon rather than somewhere else. I'm really wondering what in the enzyme's shape makes the OH added to the beta carbon, rather than somewhere else. In other words, what is the structural mechanism behind substrate specificity?

enter image description here

  • $\begingroup$ Maybe I'm misunderstanding, but the product obviously has to be epinephrine, so the enzyme will add the OH to that specific place that results in the right product. If you're asking how the enzyme does that, you might want to be more specific about which parts of the mechanism you're interested in. $\endgroup$ May 6 '12 at 19:02
  • $\begingroup$ Evolutionarily speaking - yes - the enzyme has evolved to add OH to that specific place. I'm really wondering what in the enzyme's shape makes the OH added to the beta carbon, rather than somewhere else. I'll just edit the question to include that now. $\endgroup$ May 6 '12 at 21:10
  • $\begingroup$ It's still not clear what you're asking. At the moment, I would answer: "The enzyme has evolved to do that and the body has evolved to use that molecule. That's just how it is, for some reason that exact molecule was convenient." Or are you asking about the reaction mechanism of the active site? In that case you should say that. $\endgroup$
    – Armatus
    May 6 '12 at 21:30
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    $\begingroup$ I think he is asking about what is the structural mechanism behind substrate specificity. $\endgroup$
    – bobthejoe
    May 7 '12 at 19:10
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    $\begingroup$ May be a good start is at BRENDA database brenda-enzymes.info/php/result_flat.php4?ecno= $\endgroup$
    – friveroll
    Jun 8 '12 at 18:39

There are various studies on the mechanism of action of dopamine beta-hydroxylase (DBH).

This review (sorry, non-free article) by Kaufman and Friedman was written less than 10 years after the discovery of the reaction of conversion of tyrosine into [nor]adrenaline in the adrenal gland:

DOPAMINE BETA-HYDROXYLASE - Kaufman S., Friedman S. - Pharmacol Rev. 1965 Jun;17:71-100.

It makes for an interesting read, as they start detailing the history of our knowledge on adrenaline synthesis (well, at least at that point in time!), the various hypotheses and models that were thought of, experiments with radioactive labeled precursors and so on.

They then procede to talk about the mechanism of the enzyme. Without going too much in detail, they report experiments showing that oxygen, ATP and Mg++ stimulate dopamine beta-hydroxylase enzymatic activity, with the following note on Mg++:

[...] Mg++ is required for the uptake of catecholamines by the adrenal vesicles. EDTA inhibits the uptake of the precursor, hydroxytyramine, but does not inhibit the enzymatic conversion to norepinephrine if the vesicles are intact.

They also list the requirement for two cofactors: ascorbate and copper

The first indication that Dopamine-beta-hydroxylase is a mixed-function oxidase was the demonstration of a requirement for ascorbate. It was found that this reducing agent could stimulate the hydroxylation reaction even in adrenal particles, and the requirement for ascorbate became more pronounced as the enzyme was purified.

Bringing to the reaction:

Dopamine + ascorbate + O2 → L-norepinephrine + dehydroascorbate + H2O

For the Cu:

Only recently, however, was the enzyme obtained in a pure state and the metal characterized by direct analysis. The metal is copper and it is present in a concentration of 0.65 to 1.0 ug per mg enzyme (4 to 7 moles of copper per mole of protein). It can be removed by treatment with concentrated potassium cyanide. This copper-free enzyme is inactive but can be reactivated by addition of Cu.

They then proceed to detail some basic mechanism of action, which I will not report here for briefness.

A few years later, researchers from the same group authored a book chapter for the American Chemical Society entitled:

The Mechanism of Action of Dopamine β-Hydroxylase - Kaufman S., Bridgers W.F., Baron J - in Oxidation of Organic Compounds, Chapter 73, pp 172–176, 1968 - Chapter DOI: 10.1021/ba-1968-0077.ch073

From the abstract (I do not have access to the full text of this):

Dopamine β-hydroxylase catalyzes the side-chain hydroxylation of dopamine and other phenylethylamine derivatives. Ascorbic acid serves as a specific electron-donating cofactor. The enzyme from bovine adrenal glands contains Cu2+ and a smaller amount of Cu+. When the enzyme oxidizes ascorbate to dehydroascorbate, most of the Cu2+ is reduced to Cu+. Added substrate is hydroxylated, and Cu+ is reoxidized to Cu2+.

To get to some more recent years...

In this review

New insights into copper monooxygenases and peptide amidation: structure, mechanism and function. - Prigge ST, Mains RE, Eipper BA, Amzel LM. - Cell Mol Life Sci. 2000 Aug;57(8-9):1236-59.

A mechanism for Peptidylglycine alpha-hydroxylating monooxygenase (PHM; EC is proposed, and

Since PHM is homologous in sequence and mechanism to dopamine beta-monooxygenase (DBM; EC, the enzyme that converts dopamine to norepinephrine during catecholamine biosynthesis, these structural and mechanistic insights are extended to DBM.

DBM mechanism

A structure of PHM along with a modeling of DBH is shown in Fig 14. You can see the two catalytic copper atoms and the possible binding of dopamine to the site.

PHM structure and DBH model

They also show a mechanism of action for PHM. Essentially, through the oxydation of ascorbate the Cu++ becomes Cu+ and can then bind an oxygen molecule that will be used to hydroxylate the product:

PHM mechanism

Finally, a detailed computational model of the enzyme, and its active site is shown in this paper:

Structural Insight of Dopamine β-Hydroxylase, a Drug Target for Complex Traits, and Functional Significance of Exonic Single Nucleotide Polymorphisms - Kapoor A, Shandilya M, Kundu S - PLoS One. 2011;6(10):e26509. Epub 2011 Oct 20.

The active site is shown in Fig. 7, where you can clearly see the two copper atoms in purple and green, the most important aminoacids at the active site and two water molecules in red:

Active site of DBH http://www.plosone.org/article/fetchObject.action?uri=info%3Adoi%2F10.1371%2Fjournal.pone.0026509.g007&representation=PNG_M

Figure 10C shows the interaction of dopamine with the aa of the active site (Fig 10A and B show superposition between rat and human enzymes and Fig 10D shows the interaction of a drug with the enzyme):

Residues important for catalysis http://www.plosone.org/article/fetchObject.action?uri=info%3Adoi%2F10.1371%2Fjournal.pone.0026509.g010&representation=PNG_M

Finally, a remark on your question. You say:

Evolutionarily speaking - the enzyme has evolved to add OH to the beta-carbon rather than somewhere else.

As I said somewhere else: not everything has an evolutionary explication, and evolution is not finalistic. This specific enzyme structure appeared at some point and it was evidently working fairly well, and at the time the rest of the pathway may or may not have been like it is at present, but the important thing is that the enzymatic network managed to work together. It could had well evolved to hydroxylate another position, but probably in that case we would have different enzymes downstream. The important thing to remember is that the enzyme did NOT evolve with the finality of oxydating the beta carbon.

  • $\begingroup$ great answer nico, and excellent fair-use quoting to help those without access to the article. $\endgroup$ Jun 9 '12 at 11:03
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    $\begingroup$ As an aside, I think it's crazy that copyright covers scientific articles for this long - I think copyright expiry should be much shorter for papers. $\endgroup$ Jun 9 '12 at 11:05
  • $\begingroup$ @Richard Smith: that is very true, and it makes life very difficult for researchers sometimes... (anyway, luckily the last two articles I quoted are free, the last one is under CC license). $\endgroup$
    – nico
    Jun 9 '12 at 11:27

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