Cryocrystallography and microspectrophotometry of a mutant (alpha D60N) tryptophan synthase alpha 2 beta 2 complex reveals allosteric roles of alpha Asp60.
We have investigated the role of Asp60 of the alpha-subunit in allosteric communication between the tryptophan synthase alpha- and beta-subunits. Crystallographic and microspectrophotometric studies have been carried out on a mutant (alpha D60N) tryptophan synthase alpha 2 beta 2 complex which has no observable alpha-activity, but has substantial beta-activity. Single-crystal polarized absorption spectra indicate that the external aldimine is the predominant L-serine intermediate and that the amount of the intermediate formed is independent of pH, monovalent cations, and allosteric effectors. The three-dimensional structure is reported for this mutant enzyme complexed with indole 3-propanol phosphate bound to the alpha-site and L-serine bound to the beta-site (alpha D60N-IPP-Ser), and this structure is compared with that of the unliganded mutant enzyme (alpha D60N). In the complex, L-serine forms a stable external aldimine with the pyridoxal phosphate coenzyme at the active site of the beta-subunit. The conformation of the unliganded mutant is almost identical to that of the wild type enzyme. However, the structure of the mutant complexed with IPP and serine exhibits ligand-induced conformational changes much smaller than those observed previously for another mutant enzyme in the presence of the same ligands (beta K87T-IPP-Ser) [Rhee, S., Parris, K. D., Hyde, C. C., Ahmed, S. A., Miles, E. W., and Davies, D. R. (1997) Biochemistry 36, 7664-7680]. The alpha D60N-IPP-Ser alpha 2 beta 2 complex does not undergo the following ligand-induced conformational changes: (1) the closure of the alpha-subunit loop 6 (residues 178-191), (2) the movement of the mobile subdomain (residues 93-189) of the beta-subunit, and (3) the rotation of the alpha-subunit relative to the beta-subunit. These observations show that alpha Asp60 plays important roles in the closure of loop 6 and in allosteric communication between the alpha- and beta-subunits.
Rhee
S
S
Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland 20892, USA.
Miles
E W
EW
Mozzarelli
A
A
Davies
D R
DR
D000494:Allosteric Regulation / Q000235:genetics; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D001216:Asparagine / Q000235:genetics*; D001224:Aspartic Acid / Q000235:genetics* / Q000378:metabolism*; D002384:Catalysis; D003460:Crystallization; D018360:Crystallography, X-Ray; D005615:Freezing; D007097:Imines / Q000378:metabolism; D007211:Indoles / Q000378:metabolism; D015591:Microspectrophotometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011485:Protein Binding / Q000235:genetics; D017433:Protein Structure, Secondary; D012694:Serine / Q000737:chemistry; D014367:Tryptophan Synthase / Q000737:chemistry* / Q000235:genetics*
D007097:Imines; D007211:Indoles; D001224:Aspartic Acid; C041272:indolepropanol phosphate; D012694:Serine; D001216:Asparagine; D014367:Tryptophan Synthase