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Dear experts,

I have a puzzling Q which I am unable to resolve.
I am researching a very interesting enzyme system which has
an essential  2,4,5-trihydroxyphenylalanine quinone cofactor.
This cofactor has a lambda max at 480nm and a red colour.
Extensive research has been conducted on these enzymes
(check my web page)
Anyways I am researching the enzyme mechanism
using site directed mutagenesis
I have made a bunch of mutants
all of which effect the spectral properties of the enzymes.

The most drastic change in lambda max occurred
when I replaced the essential Asp residue
(carboxyl group) with a Ala residue (methyl group).
This shifted the lambda max to 445 nm and turned the colour yellow.
Replacement of the carboxyl with the isostructural amide group (Asn
residue)
resulted in a shift to 465 nm and an orange colour.

My problem is how to go about explaining these changes.
the blue shifts are due to less intense pi-pi* transitions,
which is indicative of a reduced -ve charge on the ring?
Model studies show that deprotonation of the O4 oxygen
causes a yellow to red shift (pka ~4)
but based on the crystal structures of the mutant enzymes
this O4 still seems to be ionised.
Is it plausible to rationilise the lambda max shift
in terms of electrostatic coupling?
The shift is apparent throughout the pH range 5-9
and the cofactor is still able to bind substrate analogues,
but at a much reduced rate.

any ideas, suggestions, references, most welcome

J

--
Jeremy Murray                       MAIL:nospambmbjmm@bmb.leeds.ac.uk
Biochemistry & Molecular Biology         TEL:  +44 113 233 2591
Leeds University, LEEDS LS2 9JT          FAX:  +44 113 233 3167
        http://www.smb.leeds.ac.uk/wwwsb/jez.html
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