Probable involvement of sulfhydryl groups and a metal as essential components of the cellulase of Clostridium thermocellum

The crude extracellular cellulase from Clostridium thermocellum was oxidatively inactivated by air and inhibited by sulfhydryl reagents. Activity-loss was prevented and reversed by the addition of a high concentration (10 mM) dithiothreitol (DDT) at zero time and up to 24 h respectively. In the presence of a low concentration (0.4 mM) of DTT, the enzyme was more rapidly inactivated than in air alone. This was probably due to autoxidation of the low DTT concentration to H₂O₂ as shown by its prevention by a high DTT concentration, exclusion of air, or catalase; and by the oxidative inactivation of the enzyme by H₂O₂. The inactivation by H₂O₂ could be prevented by a high concentration of DTT but not by air exclusion. EDTA protected the enzyme from inactivation in air by a low concentration of DTT or by H₂O₂. This is presumably due to the role of metals in oxidation of SH groups. Furthermore, copper (5 M) also caused inactivation and this was prevented by the presence of a high DTT concentration. Even in the protective atmosphere of a high DTT concentration, cellulase was inactivated by certain apolar chelating agents such as o-phenanthroline and -¹-dipyridyl, such inactivation being preventable by the prior incubation of the chelator with a mixture of Fe²⁺ and Fe³⁺. These data suggest that the clostridial cellulase, unlike the enzyme from aerobic fungi, contains essential sulfhydryl groups and is stimulated by iron. The endo--glucanase component of the cellulase complex was not susceptible to oxidative inactivation.Abbreviations DTT dithiothreitol - CMC carboxymethylcellulose - DTNB 5,5-dithiobis-(2-nitrobenzoic acid) - NEM N-ethylmaleimide - p-CMB p-chloromercuribenzoic acid