Evidence that two zinc fingers in the methionine aminopeptidase from Saccharomyces cerevisiae are important for normal growth.
Limited proteolysis of intact yeast methionine aminopeptidase (MAP1) with trypsin releases a 34 kDa fragment whose NH2-terminal sequence begins at Asp70, immediately following Lys69. These results suggest that yeast MAP may have a two-domain structure consisting of an NH2-terminal zinc finger domain and a C-terminal catalytic domain. To test this, a mutant MAP lacking residues 2-69 was generated, overexpressed, purified and analyzed. Metal ion analyses indicate that 1 mol of wild-type yeast MAP contains 2 mol of zinc ions and at least 1 mol of cobalt ion, whereas 1 mol of the truncated MAP lacking the putative zinc fingers contains only a trace amount of zinc ions but still contains one mole of cobalt ion. These results suggest that the two zinc ions observed in the native yeast MAP are located at the Cys/His rich region and the cobalt ion is located in the catalytic domain. The kcat and Km values of the purified truncated MAP are similar to those of the wild-type MAP when measured with peptide substrates in vitro and it appears to be as active as the wild-type MAP in vivo. However, the truncated MAP is significantly less effective in rescuing the slow growth phenotype of map mutant than the wild-type MAP. These findings suggest that the zinc fingers are essential for normal MAP function in vivo, even though the in vitro enzyme assays indicate that they are not involved in catalysis.(ABSTRACT TRUNCATED AT 250 WORDS)
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Edward A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, MO 63104.
Guo
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Ling
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Chang
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D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002918:Chymotrypsin / Q000494:pharmacology; D003035:Cobalt / Q000032:analysis; D063208:Methionyl Aminopeptidases; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development*; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D014357:Trypsin / Q000494:pharmacology; D015032:Zinc / Q000032:analysis; D016335:Zinc Fingers / Q000235:genetics / Q000502:physiology*
D003035:Cobalt; D000626:Aminopeptidases; D063208:Methionyl Aminopeptidases; D002918:Chymotrypsin; D014357:Trypsin; D015032:Zinc