The location of the calcium ion binding site in bovine alpha-trypsin and beta-trypsin using lanthanide ion probes.
The effect of Gd3+ on the nuclear magnetic resonance (NMR) relaxation rates, T1m-1 and T2m-1, of inhibitor protons in metal-inhibitor-trypsin ternary complexes has been measured. The Solomon-Bloembergen equations have been used to calculate distances of 10.0 +/- 0.5, 8.8 +/- 0.5, and 9.5 +/- 0.5 A between the metal ion and the methyl and ortho protons of p-toluamidine, and the methyl protons of acetamidine, respectively. Essentially the same results are obtained for both alpha-trypsin and beta-trypsin. Binding constants of 3.3 x 10(3) and 4.1 x 10(3) M-1 for the association of Gd(III) with alpha-trypsin and beta-trypsin, respectively, in the presence of p-toluamidine at pH 6.0 have been obtained by equilibrium dialysis. Calcium binding constants of 260 and 3700 M-1 at pH 6.0 and 8.0, respectively, with beta-trypsin have also been obtained. Calcium ion and gadolinium ion compete for the same site on the protein. Calcium has been shown to protect alpha-trypsin from further autolytic degradation to psi-trypsin. From examination of the crystal structure of the enzyme we propose that the calcium ion binding site of bovine trypsin is comprised of the side chains of Asp-194 and Ser-190 (based on the chymotrypsin sequence numbering system). This seems to be the only site which is comprised of at least one carboxyl group; which fits our distance requirements and which is conisistent with other chemical data.
D000818:Animals; D001665:Binding Sites; D002118:Calcium; D002417:Cattle; D005682:Gadolinium; D007527:Isoenzymes; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008433:Mathematics; D010179:Pancreas / Q000201:enzymology; D011485:Protein Binding; D011487:Protein Conformation; D013696:Temperature; D014357:Trypsin
D007527:Isoenzymes; D005682:Gadolinium; D014357:Trypsin; D002118:Calcium