A dominant negative mutation in Saccharomyces cerevisiae methionine 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. In Escherichia coli MetAP, 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 yeast Saccharomyces cerevisiae MetAP1 is 41% identical to E. coli MetAP and contains these cobalt coordinating residues. Using site-directed mutagenesis on the gene coding for yeast MetAP1, we replaced Asp219 (corresponding to Asp97 in E. coli MetAP) with Asn. The yeast D219N mutant enzyme has 10(3)-fold lower catalytic activity and a different substrate specificity when compared to wild-type yeast MetAP1. These results indicate that the side-chain of Asp219 is 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 of S. cerevisiae MetAP2.
Klinkenberg
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M
School of Medicine, Saint Louis University Health Sciences Center, 1402 South Grand Boulevard, St. Louis, Missouri 63104, USA.
Ling
C
C
Chang
Y H
YH
D000626:Aminopeptidases / Q000235:genetics* / Q000378:metabolism*; D001224:Aspartic Acid / Q000235:genetics; D001665:Binding Sites; D003035:Cobalt / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005800:Genes, Fungal; D007700:Kinetics; D008666:Metalloendopeptidases / Q000378:metabolism*; D063208:Methionyl Aminopeptidases; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D013379:Substrate Specificity
D011994:Recombinant Proteins; D001224:Aspartic Acid; D003035:Cobalt; D000626:Aminopeptidases; D063208:Methionyl Aminopeptidases; C106541:methionine aminopeptidase 2; D008666:Metalloendopeptidases