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 from Bacillus subtilis and Bacillus 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 by KRAUT 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.

Bitter flavour in dairy products. II. A review of bitter peptides from caseins: their formation, isolation and identification, structure masking and inhibition

“…Proteases compatible with the substrates cou Id thus be used. Choosing alcalase, a protease known to cleave proteins at the carboxylic side of hydrophobic amino acids, would result in less bitterness th an if the cleavage were carried out by a protease c1eaving at hydrophilic amino acids (Svendsen, 1976). Thus, according to Pé-lissier and Manchon (1976), Adler-Nissen (1986c) has elaborated on the influence of the protease used for the production of enzymic hydrolysates on the development of bitterness.…”

Serine carboxypeptidases. A review

“…X-ray studies of the complexes between trypsin and pancreatic trypsin inhibitor (83) have indicated that the side chains of the amino acid residues in the P] and P' _, positions of both inhibitors interact with the enzyme but in a less specific manner than observed on the other side of the scissile bond. However, kinetic studies of several serine endopeptidases have provided evidence for the importance of interactions between enzyme and the side-chains on both sides of the scissile bond as reviewed by SVENDSEN (178). The binding sites of proteolytic enzymes determine their specificity but they probably also function to stabilize the transition state configuration.…”

“…the side-chains in chymotrypsin which form the hydrophobic crevice for the side-chain in the P, position of the substrate. While chemical modifications of essential amino acid residues usually abolish the catalytic activity, modifications of side-chains at binding sites may have different effects on activity, depending on the nature of the reagent and the importance of the particular side-chain as previously demonstrated with some proteolytic enzymes (44,80,178). Recently, it has become possible by site-directed mutagenesis to specifically exchange amino acid residues in proteins (206).…”

Influence of guanidine derivatives on the specificity of malt carboxypeptidase

“…The binding sites of proteolytic enzymes determine their specificity and chemical modifications of amino acid residues in these sites have provided information about enzyme-substrate interactions for such enzymes as subtilisin (20), thermolysin (1) and carboxypeptidase Y (4). In the latter case with interesting applications as a consequence (5,6).…”