CHARACTERIZATION OF CHITIN SYNTHASE-2 OF SACCHAROMYCES-CEREVISIAE - IMPLICATION OF 2 HIGHLY CONSERVED DOMAINS AS POSSIBLE CATALYTIC SITES

Chitin synthase 2 of Saccharomyces cerevisiae was characterized by means of site-directed mutagenesis and subsequent expression of the mutant enzymes in yeast cells. Chitin synthase 2 shares a region whose sequence is highly conserved in all chitin synthases. Substitutions of conserved amino acids in this region with alanine (alanine scanning) identified two domains in which any conserved amino acid could not be replaced by alanine to retain enzyme activity. These two domains contained unique sequences, Glu(561)-Asp(562)-Arg(563) and Gln(601)-Arg(602)-Arg-(603)-Arg(604)-Trp(605), that were conserved in all types of chitin synthases. Glu(561) or arginine at 563, 602, and 603 could be substituted by glutamic acid and lysine, respectively, without significant loss of enzyme activity. However, even conservative substitutions of Asp(562) with glutamic acid, Gln(601) with asparagine, Arg(604) with lysine, or Trp(605) with tyrosine drastically decreased the activity, but did not affect apparent K-m values for the substrate significantly. In addition to these amino acids, Asp(441) Was also found in all chitin synthase. The mutant harboring a glutamic acid substitution for Asp(441) severely lost activity, but it showed a similar apparent K-m value for the substrate, Amounts of the mutant enzymes in total membranes were more or less the same as found in the wild type. Furthermore, Asp(441), Asp(562), Gln(601), Arg(604), and Trp(605) are completely conserved in other proteins possessing N-acetylglucosaminyltransferase activity such as NodC proteins of Rhizobium bacterias. These results suggest that Asp(441), Asp(562), Gln(601), Arg(604),,and Trp(605) are located in the active pocket and that they function as the catalytic residues of the enzyme.