Study on the characteristics of the reaction mode and the thermostability of THERMOLYSIN

Abstract

Thermolysin is a heat-stable endopeptidase isolated from Bacillus thermoproteolyticus. The enzyme binds one zinc ion at the active site and four calcium ions at three distinct sites on the enzyme surface. In this paper, we investigated the specificity of thermolysin for substrates, the effect of pH and temperature on the enzyme activity and the effect of calcium ions on the thermostability of the protein. In the study, degree of the enzyme reaction was measured by the spectrophotometrical ninhydin reaction method, and the thermal behavior of the enzyme was monitered spectrophotometrically by using the change of optical density of the enzyme solution according to the elivation of temperature which must be associated with the conformational change of the protein. In the study on the substrate specificity, it was found that the R$_1$, side chain is a critial site and the peptide bond on the imino side of the bulky hydro phobic amino acid residue is effectively hydrolyzed, and also the hydrophobicity of the R1 side chain promotes the hydrolysis of the scissle peptide bond. The optimal pH range was between 7.0 and 7.5 and the pH profile was a bell-shape. The enzyme activity increased as temperature elivated from a low temperature ($25\,^\circ\!C$) to $80\,^\circ\!C$. The activation energy was 4.4 kcal/mole at pH 7.5. In the study on the effect of calcium ions on the thermal stability of the enzyme, the melting temperature(Tm) increased from $77\,^\circ\!C$ to about $90\,^\circ\!C$ as the calcium ion concentration increased from below $10^{-5}$M to $10^{-2}$M. And the Tm value decreased to about $50\,^\circ\!C$ in 10 mM EDAT, the metal ion chelating agent, solution, but in 2 mM phenanthroline, the chelating agent specific to the zinc ion, solution, the melting profile of the protein was same as in the solution of the low calcium ion concentration (below $10^{-5}$M). Based on a series of these experiments it was proposed that the calcium ion binding to thermolysin is ...