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Cryocrystallography and microspectrophotometry of a mutant (alpha D60N) tryptophan synthase alpha 2 beta 2 complex reveals allosteric roles of alpha Asp60.
Biochemistry. 1998 Jul 28; 37(30):10653-9.B

Abstract

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.

Authors+Show Affiliations

Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland 20892, USA.No affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

9692955

Citation

Rhee, S, et al. "Cryocrystallography and Microspectrophotometry of a Mutant (alpha D60N) Tryptophan Synthase Alpha 2 Beta 2 Complex Reveals Allosteric Roles of Alpha Asp60." Biochemistry, vol. 37, no. 30, 1998, pp. 10653-9.
Rhee S, Miles EW, Mozzarelli A, et al. Cryocrystallography and microspectrophotometry of a mutant (alpha D60N) tryptophan synthase alpha 2 beta 2 complex reveals allosteric roles of alpha Asp60. Biochemistry. 1998;37(30):10653-9.
Rhee, S., Miles, E. W., Mozzarelli, A., & Davies, D. R. (1998). Cryocrystallography and microspectrophotometry of a mutant (alpha D60N) tryptophan synthase alpha 2 beta 2 complex reveals allosteric roles of alpha Asp60. Biochemistry, 37(30), 10653-9.
Rhee S, et al. Cryocrystallography and Microspectrophotometry of a Mutant (alpha D60N) Tryptophan Synthase Alpha 2 Beta 2 Complex Reveals Allosteric Roles of Alpha Asp60. Biochemistry. 1998 Jul 28;37(30):10653-9. PubMed PMID: 9692955.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Cryocrystallography and microspectrophotometry of a mutant (alpha D60N) tryptophan synthase alpha 2 beta 2 complex reveals allosteric roles of alpha Asp60. AU - Rhee,S, AU - Miles,E W, AU - Mozzarelli,A, AU - Davies,D R, PY - 1998/8/7/pubmed PY - 1998/8/7/medline PY - 1998/8/7/entrez SP - 10653 EP - 9 JF - Biochemistry JO - Biochemistry VL - 37 IS - 30 N2 - 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. SN - 0006-2960 UR - https://www.unboundmedicine.com/medline/citation/9692955/Cryocrystallography_and_microspectrophotometry_of_a_mutant__alpha_D60N__tryptophan_synthase_alpha_2_beta_2_complex_reveals_allosteric_roles_of_alpha_Asp60_ L2 - https://doi.org/10.1021/bi980779d DB - PRIME DP - Unbound Medicine ER -