Jeremy Murray <bmbjmm at bmb.leeds.ac.uk> wrote in article
<367BF9C7.F5491FD9 at bmb.leeds.ac.uk>...
> 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
> 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
See you are at Leeds...so was I.
Suggest you might take a walk up University Road,Clothworkers Court,Dept of
Colour Chemistry and see if you can talk to Dr John Griffiths who is well
known for his work on the Colour and Constitution of Organic Molecules.