CHARACTERIZATION OF CHITIN SYNTHASE-2 OF SACCHAROMYCES-CEREVISIAE .2.BOTH FULL-SIZE AND PROCESSED ENZYMES ARE ACTIVE FOR CHITIN SYNTHESIS

When chitin synthase 2 of Saccharomyces cerevisiae was overexpressed i n yeast cells using GAL1 promoter, deletion of the N-terminal 193 amin o acids significantly increased the level of the protein without affec ting its characteristics. We partially purified N-terminally truncated chitin synthase 2 by product entrapment and ion exchange column chrom atography, and found that it was active even without trypsin treatment when appropriate divalent cations were present in the reaction mixtur e. This chitin synthase activity was independent of the N-terminal 193 amino acid truncation, because partially purified full length enzyme also exhibited the activity without trypsin treatment in the presence of appropriate cations. Furthermore, the molecular weights of these tw o forms of chitin synthase 2 were coincident with those estimated from the deduced amino acid sequence, and most of the chitin synthase 2 in the yeast membrane was present as an unprocessed form, as judged from its molecular weight. Treatment of either full length or truncated en zyme with trypsin. however, further increased the enzyme activity by f our to fivefold, and produced a 35 kDa polypeptide that specifically r eacted with monoclonal antibody raised against the region containing t he putative active site of chitin synthase 2. Thus, it appears that pr edominant native (unprocessed) chitin synthase 2 is active, but the 35 kDa region encompassing the active site is sufficient for the catalyt ic activity.