A SPECTROPHOTOMETRIC STUDY ON THE INTERACTION OF THERMOLYSIN WITH CHLORIDE AND BROMIDE IONS, AND THE STATE OF TRYPTOPHYL RESIDUE-115

The activity of thermolysin is greatly enhanced in the presence of hig h concentrations of neutral salts [Holmquist, B. and Vallee, B.L. (197 6) Biochemistry 15, 101-107; Inouye, K. (1992) J. Biochem. 112, 335-34 0]. NaBr and NaCl are the most effective for the activation. An absorp tion difference spectrum with a peak around 293 nm, which is character istic of the red-shift of a tryptophyl residue caused by charge effect s, was observed on mixing of thermolysin with NaCl. As the peak disapp eared in the presence of competitive inhibitors of the enzyme (phospho ramidon and zincov), it was considered to be derived from a tryptophyl residue (Trp 115) located in the active site of the enzyme. On the ot her hand, this peak was not observed on the mixing of thermolysin and NaBr, indicating that the slight difference in size between chloride a nd bromide ions is critical for the interaction with the tryptophyl re sidue. NaCl and NaBr exhibit comparable effects on the activation of t hermolysin regardless of the considerable discrepancy in their effects on the absorptivity difference around 293 nm. This suggests that the interaction of salts with Trp 115 is not necessarily correlated with t he activation of thermolysin.