Chemical modifications of the subtilisins with special reference to the binding of large substrates. A review

Abstract

Subtilisin type Carlsberg and subtilisin BPN' are two well characterized extracellular proteases fromBacillus subtilis andBacillus amyloquefaciens, respectively. The present review summarizes the various chemical modification studies which have been performed with these enzymes. Of special interest has been those modifications which lead to changes in the enzymatic properties with regard to the hydrolysis of polypeptide substrates but not small synthetic ester substrates. In this way it is possible to obtain information about how large an area of the surface of the enzyme is involved in the binding of natural substrates (e.g. clupein, gelatine, casein). Nitration, iodination, glutarylation or succinylation of the subtilisins leads to increases in the hydrolyses of clupein and gelatine, while modification of carboxyl groups leads to a decrease. In all these cases the hydrolysis of small ester substrates is almost unaffected. A section of the review deals with the comparison between the results obtained by chemical modification studies and the two other methods available for the study of “secondary binding sites”: X-ray diffraction analyses and kinetic studies with synthetic polypeptides. The productive binding mode for polypeptides proposed byKraut and coworkers is in agreement with the results obtained by nitration of the subtilisins. However, results from our laboratory and the literature point towards more than one mode of productive binding. These results are discussed. Finally a comparison is made between the secondary binding sites in subtilisin and other proteolytic enzymes, especially the serine proteases. The importance of secondary interactions between enzyme and substrate is discussed.