STRUCTURAL FLEXIBILITY IN EXTREMELY STABLE CARRIER-BOUND CHYMOTRYPSIN

The binding of chymotrypsin to polyacrylamide matrices via azide coupl ing results in extreme stabilization of the enzyme towards heat, organ ic solvents or urea. About 20% of its original activity withstands the treatment at 100 degrees C for 2 h. The carrier-enzyme complex can be kept in methanol, acetone, dimethylformamide, hexane or benzene (0-99 %) for 2 h at 30 degrees C without any loss of activity. The kinetics of thermal and urea inactivation proceed in a sharply biphasic way, su ggesting that only a subpopulation of enzyme is stabilized. Several ap proaches, such as spin labelling of the active site by substrate-analo gous inhibitors, variation of the coupling procedure, chemical modific ation of the protein surface, activation from chymotrypsinogen and inh ibition by phenylmethanesulphonyl fluoride were used to characterize t he structural flexibility of the stabilized chymotrypsin as well as th e process of unfolding. The results show that carrier-enzyme interacti ons do not impair the structural flexibility necessary for activation from chymotrypsinogen or inhibition by phenylmethanesulphonyl fluoride , whereas their influence on thermal stability is strongly dependent o n the number of amino groups on the protein surface. The active site, as probed by differently-long spin-labelled oligopeptide chloromethane s, is not essentially injured by carrier binding, whereas changes in t he spectra reflecting thermal or urea-induced unfolding are much slowe r in the carrier-bound than in the soluble enzyme. Indications of two or more subpopulations of bound spin-labelled enzyme molecules differi ng in the unfolding rate are observed in thermal as well as urea denat uration. The results are discussed with regard to different models of biphasic inactivation kinetics.