The calcium ion binding site in bovine chymotrypsin A

The effect of Gd³⁺ on the nuclear magnetic resonance (nmr) relaxation rates, T_1m^?1 and T_2m^?1, of inhibitor protons in metal-inhibitor-α-chymotrypsin ternary complexes has been measured. The Solomon-Bloembergen equations were used to calculate the distance from the methyl protons of p-toluamidine (a competitive inhibitor) to the Gd³⁺ binding site which is 9.2 ± 0.5 Å. Calcium ion and gadolinium ion compete for the same binding site on α-chymotrypsin. Distances from the specificity pocket of α-chymotrypsin to the metal binding site have been measured by fluorescence energy transfer experiments. By observing energy transfer between proflavine and Nd³⁺, Pr³⁺, or Ho³⁺, we have been able to calculate a distance of approximately 10 Å between the two chromophores. This agrees well with the data obtained by nmr techniques and also gives nearly identical values to those obtained for trypsin (Darnall, D., Abbott, F., Gomez, J. E., and Birnbaum, E. R., Biochemistry15, 5017, 1976). This is consistent with the calcium ion binding sites being composed of the same residues in both trypsin and α-chymotrypsin.