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| Item type: | Article | ||||
|---|---|---|---|---|---|
| Journal or Publication Title: | Biochemistry | ||||
| Publisher: | American Chemical Society | ||||
| Number of Issue or Book Chapter: | 49 | ||||
| Page Range: | pp. 10842-10853 | ||||
| Date: | 2010 | ||||
| Institutions: | Biology, Preclinical Medicine > Institut für Biophysik und physikalische Biochemie > Prof. Dr. Reinhard Sterner | ||||
| Identification Number: |
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| Dewey Decimal Classification: | 500 Science > 570 Life sciences | ||||
| Status: | Published | ||||
| Refereed: | Yes, this version has been refereed | ||||
| Created at the University of Regensburg: | Yes | ||||
| Item ID: | 20688 |
Abstract
The prototypical tryptophan synthases form a stable heterotetrameric RββR complex in which the constituting TrpA and TrpB1 subunits activate each other in a bidirectional manner. The hyperthermophilic archaeon Sulfolobus solfataricus does not contain a TrpB1 protein but instead two members of the phylogenetically distinct family of TrpB2 proteins, which are encoded within (sTrpB2i) and ...
Abstract
The prototypical tryptophan synthases form a stable heterotetrameric RββR complex in which the
constituting TrpA and TrpB1 subunits activate each other in a bidirectional manner. The hyperthermophilic
archaeon Sulfolobus solfataricus does not contain a TrpB1 protein but instead two members of the
phylogenetically distinct family of TrpB2 proteins, which are encoded within (sTrpB2i) and outside
(sTrpB2a) the tryptophan operon. It has previously been shown that sTrpB2a does not functionally or
structurally interact with sTrpA, whereas sTrpB2i substantially activates sTrpA in a unidirectional manner.
However, in the absence of catalysis, no physical complex between sTrpB2i and sTrpA could be detected. In
order to elucidate the structural requirements for complex formation, we have analyzed the interaction
between sTrpA (R-monomer) and sTrpB2i (ββ-dimer) by means of spectroscopy, analytical gel filtration, and
analytical ultracentrifugation, as well as isothermal titration calorimetry. In the presence of the TrpA ligand
glycerol 3-phosphate (GP) and the TrpB substrate L-serine, sTrpA and sTrpB2i formed a physical complex
with a thermodynamic dissociation constant of about 1 μM, indicating that the affinity between the R- and
ββ-subunits is weaker by at least 1 order of magnitude than the affinity between the corresponding subunits of
prototypical tryptophan synthases. The observed stoichiometry of the complex was 1 subunit of sTrpA per 2
subunits of sTrpB2i, which corresponds to a Rββ quaternary structure and testifies to a strong negative
cooperativity for the binding of the R-monomers to the ββ-dimer. The analysis of the interaction between
sTrpB2i and sTrpA in the presence of several substrate, transition state, and product analogues suggests that
the Rββ complex remains stable during the whole catalytic cycle and disintegrates into R- and ββ-subunits
upon the release of the reaction product tryptophan. The formation of a transient tryptophan synthase
complex, together with the observed low affinity of sTrpB2i for L-serine, couples the rate of tryptophan
biosynthesis in S. solfataricus to the cytosolic availability of L-serine.
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