ISSN:
0887-3585
Keywords:
staphylococcal nuclease
;
mechanism of
;
ternary enzyme-La3+-dTdA complex
;
active site
;
trinucleotide complex of
;
assignments of 1H aromatic resonances
;
assignments of 15N resonances
;
HMQC studies of
;
NOESY-HMQC studies of
;
energy minimization of
;
Chemistry
;
Biochemistry and Biotechnology
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Medicine
Notes:
The conformation of the staphylococcal nuclease-bound metal-dTdA complex, previously determined by NMR methods [Weber, D.J., Mullen, G.P., Mildvan, A.S. (1991) Biochemistry 30:7425-7437] was docked into the X-ray structure of the enzyme-Ca2+-3′,5′-pdTp complex [Loll, P.J., Lattman, E.E. (1989) Proteins: Struct., Funct., Genet. 5:183-201] by superimposing the metal ions, taking into account intermolecular nuclear Overhauser effects from assigned aromatic proton resonances of Tyr-85, Tyr-113, and Tyr-115 to proton resonances of the leaving dA moiety of dTdA, and energy minimization to relieve small overlaps. The proton resonances of the Phe, Tyr, and Trp residues of the enzyme in the ternary enzyme-La3+-dTdA complex were sequence specifically assigned by 2D phase-sensitive NOESY, with and without deuteration of the aromatic protons of the Tyr residues, and by 2D heteronu-clear multiple quantum correlation (HMQC) spectroscopy and 3D NOESY-HMQC spectros-copy with 15N labeling. While resonances of most Phe, Tyr and Trp residues were unshifted by the substrate dTdA from those found in the enzyme-La3+-3′,5′-pdTp complex and the enzyme-Ca2+-3′,5′-pdTp complex, proton resonances of Tyr-85, Tyr-113, Tyr-115, and Phe-34 were shifted by 0.08 to 0.33 ppm and the 15N resonance of Tyr-113 was shifted by 2.1 ppm by the presence of substrate. The optimized position of enzyme-bound dTdA shows the 5′-dA leaving group to partially overlap the inhibitor, 3′,5′-pdTp (in the X-ray structure). Tne 3′-TMP moiety of dTdA points toward the solvent in a channel defined by Ile-18, Asp-19, Thr-22, Lys-45, and His-46. The phosphate of dTdA is coordinated by the metal, and an adjacent inner sphere water ligand is positioned to donate a hydrogen bond to the general base Glu-43 and to attack the phosphorus with inversion. Arg-35 and Arg-87 donate monodentate hydrogen bonds to different phosphate oxygens of dTdA, with Arg-87 positioned to protonate the leaving 5′-oxygen of dA, thus clarifying the mechanism of hydrolysis. Model building of an additional 5′-dGMP onto the 3′-oxygen of dA placed this third nucleotide onto a surface cleft near residues Glu-80, Asp-83, Lys-84, and Tyr-115 with its 3′-OH group accessible to the solvent, thus defining the size of the substrate binding site as accommodating a trinucleotide. © 1992 Wiley-Liss, Inc.
Additional Material:
9 Ill.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1002/prot.340130402
