Dynamic and structural properties of the calcium binding site of bovine serine proteases and their zymogens. A multinuclear nuclear magnetic resonance and stopped-flow study.
The combined use of 43Ca and 113Cd nuclear magnetic resonance (n.m.r.) has provided information on the structural and dynamic properties of the calcium binding site located in homologous positions in bovine beta-trypsin, alpha-chymotrypsin and their zymogens. The 43Ca and 113Cd n.m.r. chemical shifts are consistent with an octahedral symmetry of the binding site and with the substitution of one of the two carboxylate ligands present in trypsin(ogen) with a neutral ligand in chymotrypsin(ogen). The constancy of the 113Cd n.m.r. chemical shifts upon binding of the pancreatic trypsin inhibitor and/or the dipeptide Ile-Val to trypsinogen confirms that structural changes in the activation domain do not affect the calcium binding site. The exchange between bound and "free" (solvated) Ca2+ is slow on the 43Ca n.m.r. time-scale for trypsin(ogen), but falls in the intermediate exchange region for chymotrypsin(ogen). In trypsin, the Ca2+ off-rate was measured by stopped-flow making use of the calcium indicator 1,2-bis(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid and was found to be 3(+/- 1) s-1. In chymotrypsin(ogen) the off-rates calculated from the 43Ca n.m.r. data are 70 s-1 and 350 s-1, respectively. The dynamic properties of the calcium binding site of serine (pro)enzymes have been related to the flexibility of the binding site itself and have been compared to those of other extracellular and intracellular calcium binding proteins.
D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002417:Cattle; D002918:Chymotrypsin; D002919:Chymotrypsinogen; D003594:Cytoplasmic Granules; D010450:Endopeptidases; D004792:Enzyme Precursors; D009682:Magnetic Resonance Spectroscopy; D012697:Serine Endopeptidases; D014357:Trypsin; D014362:Trypsinogen
D004792:Enzyme Precursors; D014362:Trypsinogen; D002919:Chymotrypsinogen; D010450:Endopeptidases; D012697:Serine Endopeptidases; D002918:Chymotrypsin; D014357:Trypsin; D002118:Calcium