title: Multinuclear magnetic resonance studies on the calcium (II) binding site in trypsin, chymotrypsin, and subtilisin.
abstract: Two different nuclear magnetic resonance experiments were conducted to elucidate the properties of the Ca(II) binding locus on serine proteases in solution. Trypsin, alpha-chymotrypsin, and subtilisin were inactivated with diisopropyl fluorophosphate, and the distance of the phosphorus from Gd(III) in place of Ca(II) was determined from the lanthanide-induced relaxation on the 31P resonance. The distances found (between 20 and 21 A) were in excellent agreement with those reported in the X-ray crystallographic structures of trypsin and subtilisin, demonstrating that the method has wide applicability to systems for which no X-ray structure is available. Subsequently, the 113Cd spectra [in place of Ca(II)] were examined in the presence of the native enzymes. At ambient temperatures only a single 113Cd resonance could be observed, presumably representing the weighted average of the variously weakly bound ions and the free ion. At 280 K for trypsin and chymotrypsin, and at 268 K for subtilisin there was observed a resonance at ca. 65-70 ppm higher field than the previous averaged resonance that could be attributed to tightly bound Cd. The chemical shift of the resonance was consistent with its assignment to an octahedral environment around Cd with oxygen ligands.
authors.affiliation: Department of Chemistry, Rutgers, State University of New Jersey, Newark 07102.
authors.forename: F
authors.initials: F
authors.lastname: Adebodun
authors.forename: F
authors.initials: F
authors.lastname: Jordan
mesh_terms: D001665:Binding Sites; D002104:Cadmium; D002118:Calcium / Q000378:metabolism*; D002918:Chymotrypsin / Q000378:metabolism*; D005682:Gadolinium; D006863:Hydrogen-Ion Concentration; D007531:Isoflurophate; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008433:Mathematics; D008674:Metals, Rare Earth; D013381:Subtilisins / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism*
chemical_list: D008674:Metals, Rare Earth; D002104:Cadmium; D007531:Isoflurophate; D005682:Gadolinium; D013381:Subtilisins; D002918:Chymotrypsin; D014357:Trypsin; D002118:Calcium