Tryptophan Synthase:

Nature’s Nano Machine

My research is directed towards examining the structure, function, and allosteric communications of the Tryptophan Synthase enzyme.  Tryptophan synthase carries out the final two steps in the pathway to make the amino acid tryptophan.  The enzyme is quite interesting in that it uses machinelike steps to take in the first reactant at one site, cleave it, and then transfer the intermediate 2.5 nm via an interconnecting tunnel to react with the other reactant taken in at a second site.  Tryptophan synthase is used as a model system in that by studying how the different parts of the enzyme interact to carry out its precisely timed reaction, it will be possible to understand how other enzymes work and ultimately develop drugs to control their various enzymatic activities. 

The picture on the left shows a ribbon structure of tryptophan synthase and the picture on the right is a cartoon depiction of the enzyme.  The enzyme consists of two subunits, labeled a and b, which have an interconnecting tunnel between them.  When ligands are bound to the enzyme, flexible loops fold over the tunnel exits and act as lids to trap substrates inside.  The cartoon on the right shows the natural inputs:  indole glycerol phosphate (IGP) and Serine (L-Ser) and the derived outputs:  glyceraldehyde 3-phosphate (G3P) and Tryptophan (L-Trp).

A more detailed scheme of the reaction is shown in the figure below.  Here, the sequence of events in the reaction and their interactions between the subunits are presented as a map.  On the left side IGP enters and binds to the a-subunit which causes the subunit to convert to a closed form.  The conformation change sends a signal to the b-subunit to inform it that the a-subunit has bound its ligand (IGP).  On the right side, L-Ser enters the b-subunit and reacts with a pyridoxal phosphate cofactor bound to the enzyme as an internal aldimine through a linkage with bLys87.  The Ser reaction progresses through a number of intermediates and ends up as an aminoacrylate Schiff base.  The series of reactions cause a change in the b-subunit to a closed conformation, which sends an activation signal to the a-site.  The enzyme is set up so that the a-site will not cleave the indole moiety from IGP until it has received an activation signal from the b-site.  This timing element is designed to prevent the loss of indole when the b-site is not closed.  Once the a-site cleaves the indole from IGP, the indole passes through the tunnel and reacts with the aminoacrylate species generating two forms of quinonoid intermediates.  The second quinonoid then converts to an external aldimine, which causes a conformation change to the b-subunit that passes an inactivation signal to the a-site.  The external aldimine passes through one more intermediate, converts to a fully open conformation and releases L-Trp.  The a-site also converts to an open configuration and releases the G3P left over from IGP cleavage after it receives the inactivation signal from the b-subunit.  Thus, the enzyme is now re-set and ready for another set of reactions.

Many of the chemical intermediates have unique spectroscopic signatures, which allows for a method to track them during the course of a reaction.  By using rapid mixing stopped flow techniques, reaction rates for the different portions of the reaction can be detected.

My first project in the lab used an idoline derivative of tryptophan called dihydroiso-tryptophan (DIT).  This compound was shown to react in the reverse direction and release indoline as it formed aminoacrylate.  By using an IGP analog, glycerol phosphate (GP), to close the a-subunit, the indoline was shown to become trapped within the interconnecting tunnel in the enzyme (see publication for more details).  The next project I was involved with was the development of fluorinated a-site ligand compounds to be used in NMR studies.  As reactions take place at the b-site, the chemical environment at the a-site can be monitored using ¹⁹F NMR techniques to determine allosteric effects.  My current project involves examining leakage rates of indoline from the DIT reaction using different a-site ligands.

The Dunn lab has developed many compounds that can bind to the active sites of the enzyme which either inhibit activity or generate analogues of tryptophan.  The design of these compounds uses the same techniques that are used for drug design and it is through the understanding of their allosteric effects that we hope to improve the ability to predict drug activities in other enzyme systems.

Publications:

Intermediate Trapping via a Conformational Switch in the Na⁺-Activated Tryptophan Synthase Bienzyme Complex.  Harris, R. M. and Dunn, M. F.  (2002) Biochemistry 41, 9982-9990. 

The Onset of Convective Instability in a Triply Diffusive Fluid Layer.  Pearlstein, A. J., Harris, R. M., and Terrones, G.  (1989) J Fluid Mech 202, 443-465.

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