Enzymes and Autocatalysis

Achim Recktenwald, by way of Ralph Gainey <sesame at mcn.org> achim at CAM.ORG
Tue Jan 14 21:01:45 EST 1997

Sorry for not being clear. I'll go a bit more into detail.
The enzyme phenylalanine ammonia lyase (PAL) is one of the most
important and most widely available enzymes; it occurrs in all plants,
where it is the first enzyme of a major secondary pathway. The only
group, where it has not been detected yet are the animals. 

It converts phenylalanine to trans-cinnamic acid (t-CA) and ammonia.
t-CA is the starting compound for a wide range of products. It is
assumed that ~30% of all carbon incorporated into the biosphere has to
pass through PAL; e.g., all lignins, flavons, many alkaloids, etc.

HCOOH-HCNH2-CH2-phenyl <------> HCOOH-HC=CH-phenyl + NH3

The enzyme has in its active center an unusual amino acid,
dehydro-alanine (dhALA), which is 'somehow' produced by converting a
cysteine residue. This amino acid dhALA is very seldom; I do not know of
any other example than PAL. 
PAL is not an enzyme naturally occuring in E. coli. But if you clone PAL
into E. coli you obtain an active enzyme not distinguishable from the
native wild-type-plant or wt-fungal variant. It is very unlikely that E.
coli has by chance the necessary cellular machinery to concert CYS to
dhALA. Therefore, the hypotheses is  that he nascent PAL, during the
folding process to the active form auto-catalytically converts CYS to

It could theoretically be produced by removal of a water-molecule from
cysteine. But water is chemically a very bad leaving-group. The double
bond in t-cinnamic acid would be much easier achieved after prior
phosphorylation. Therefore, the sequences of a whole bunch of PAL-genes
(which by the way are highly homologous (70 - 80%) over the whole
spectrum of species from all kingdoms) have been compared with kinases.
Part of the PAL-sequence are highly homologeous to the actice-center
part of kinases.

The theory for the formation of active PAL goes therefore like this:
PAL is produced by the ribosome --> the protein or parts of it start to
fold, forming a kinase --> the kinase phosphorylates the active center
CYS in the same protein strand --> the phosphate is leaving (by its onwn
means or again through another autocatalytic process) under formation of

Writing it down makes it sound even wilder than reading it in a

There are some additional facts which distinguish this enzyme from
others, e.g.:
It is very large for this simple and small substrate; it's a  tetramer
of 78kDa subunits, has a very low activity of max. 5IU/mg = ~75nkat.
There is no known way to separate the subunits and 
re-form the active enzyme from the subunits. It has the strongest
product-inhibition I have ever encountered: the Ki for the product is
1/30 - 1/50 of the Km for the substrate, shutting it down almost as soon
as free product is available. Nobody succeded ever in deleting parts of
the sequence without complete loss of activity. Almost all
single-residue mutations produce a  dead enzyme. Because the enzyme is
present in bacteria, fungi and plants, and with sequence-homologies
close to 80%, making a second or third independent development very
unlikely, it is assumed to be ~2-3billion years old.

I copy part of Ralph's post from above:
> Are we to regard all reactions
> occurring in the interior of macromolecules as "autocatalytic"? If, as you
> hypothesize, the phosphate group participates in the [substitution?]
> reaction, could we not say that IT is the "catalyst"?

I call this reaction autocatalytic, because (if the theory is correct)
it is an intramolecular enzymatic reaction. Comparable reactions in
chemistry are also called autocatalytic.

Concerning the phophate-group: the phosphorylation would transfer energy
from ATP or some other unknown activated-compound to the CYS; the energy
released during the removal of this phosphate is used to introduce the
double-bond. So, the phosphate would be a substrate.

I hope the whole story is a bit clearer now. 
Let me know what you think of this theory, which by the way is not my
own. I read it in a PhD-Thesis. If I read correctly between the lines,
the hypothesis is studied further by subsaequent PhD-candidates.


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