A Dominant Negative Mutation inSaccharomyces cerevisiaeMethionine Aminopeptidase-1 Affects Catalysis and Interferes with the Function of Methionine Aminopeptidase-2

Methionine aminopeptidase (MetAP) enzymes require the metal ion cobalt, but little is known about the role of cobalt in the structural stability or catalysis of these enzymes. InEscherichia coliMetAP, for which a crystal structure is available, the five amino acid residues liganding the two cobalt ions are Asp97, Asp108, His171, Glu204, and Glu235. These five amino acids are conserved in all MetAPs sequenced to date. The C-terminal domain of the yeastSaccharomyces cerevisiaeMetAP1 is 41% identical toE. coliMetAP and contains these cobalt coordinating residues. Using site-directed mutagenesis on the gene coding for yeast MetAP1, we replaced Asp219(corresponding to Asp97inE. coliMetAP) with Asn. The yeast D219N mutant enzyme has 103-fold lower catalytic activity and a different substrate specificity when compared to wild-type yeast MetAP1. These results indicate that the side-chain of Asp219is important for catalysis. Expression of D219N-MetAP1 in yeast causes a slow-growth phenotype and interferes with wild-type MetAP1 in a dominant manner. Expression of D219N-MetAP1 also affects the function ofS. cerevisiaeMetAP2.