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 mea ns of site-directed mutagenesis and subsequent expression of the mutan t enzymes in yeast cells. Chitin synthase 2 shares a region whose sequ ence is highly conserved in all chitin synthases. Substitutions of con served amino acids in this region with alanine (alanine scanning) iden tified two domains in which any conserved amino acid could not be repl aced by alanine to retain enzyme activity. These two domains contained unique sequences, Glu(561)-Asp(562)-Arg(563) and Gln(601)-Arg(602)-Ar g-(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 substitu ted by glutamic acid and lysine, respectively, without significant los s of enzyme activity. However, even conservative substitutions of Asp( 562) with glutamic acid, Gln(601) with asparagine, Arg(604) with lysin e, or Trp(605) with tyrosine drastically decreased the activity, but d id not affect apparent K-m values for the substrate significantly. In addition to these amino acids, Asp(441) Was also found in all chitin s ynthase. The mutant harboring a glutamic acid substitution for Asp(441 ) severely lost activity, but it showed a similar apparent K-m value f or the substrate, Amounts of the mutant enzymes in total membranes wer e more or less the same as found in the wild type. Furthermore, Asp(44 1), Asp(562), Gln(601), Arg(604), and Trp(605) are completely conserve d in other proteins possessing N-acetylglucosaminyltransferase activit y 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.