Fluorescence properties of Savinase((R)): the X-ray structure in the region of the tryptophyl residues

Fluorescence properties of the alkaline proteinase Savinase(R) are describe d and related to the enzyme X-ray crystal structure. The intrinsic protein emission is dominated by the 'exposed' Trp 241. The fluorescence decay of t he phenylmethanesulfonyl (PMS)-proteinase excited at 297 nm was well fitted by three exponentials with lifetimes of 0.34 +/- 0.06 ns (39.4%), 2.80 +/- 0.17 ns (42.3%) and 6.80 +/- 1.15 ns (18.3%). Savinase(R) and the closely related proteolytic enzyme Esperase(R) are an attractive couple of proteins for fluorescence studies. Trp 6 and Trp 113, common to both proteinases, a re located in identical microenvironments in the two globular proteins. Iod ide ions are efficient quenchers of the PMS-Savinase(R) fluorescence. Caesi um had almost no effect on the indole emission. An electrostatic parameter E = 12.4 was obtained as the ratio K-sv(-)/K-sv(+). This means a positive m icroenvironment of the emitting tryptophans. Acrylamide quenching proceeds via both dynamic and static processes. The activation energy for the therma l deactivation of the excited indole groups is 59.2 kJ mol(-1) in the absen ce of extraneous calcium and 76.4 kJ mol(-1) in the presence of 100 mM Ca2. The T-m values in the absence and presence of added Ca2+ are 61 and 78 de grees C, respectively; saturation of the 'weak' calcium-binding site, Ca2, dramatically increased the PMS-proteinase thermostability. A good correlati on between the spectroscopic properties and the crystallographic structure of Savinase(R) was observed. (C) 1999 Elsevier Science B.V. All rights rese rved.