Identification of the catalytic residues in the double-zinc aminopeptidase from Streptomyces griseus.
The aminopeptidase from Streptomyces griseus (SGAP) has been cloned and expressed in Escherichia coli. By growing the cells in the presence of 1 M sorbitol at 18 degrees C, the protein was obtained in a soluble and active form. The amino acid sequence of the recombinant SGAP contained four amino acids differing from the previously published sequence. Re-sequencing of the native protein indicated that asparagines 70 and 184 are in fact aspartic acids as in the recombinant protein. Based on the crystal structure of SGAP, Glu131 and Tyr246 were proposed to be the catalytic residues. Replacements of Glu131 resulted in loss of activity of 4-5 orders of magnitude, consistent with Glu131 acting as the general base residue. Mutations in Tyr246 resulted in about 100-fold reduction of activity, suggesting that this residue is involved in the stabilization of the transition state intermediate.
Department of Biotechnology and Food Engineering, Institute of Catalysis Science and Technology, Technion-Israel Institute of Technology, Haifa 32000, Israel.
Yifat
Y
Fundoiano-Hershcovitz
Larisa
L
Rabinovitch
Yael
Y
Langut
Vera
V
Reiland
Gil
G
Shoham
Yuval
Y
Shoham
D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D020134:Catalytic Domain; D017931:DNA Primers; D007700:Kinetics; D007931:Leucyl Aminopeptidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D013305:Streptomyces griseus / Q000201:enzymology*
D001426:Bacterial Proteins; D017931:DNA Primers; D011994:Recombinant Proteins; D007931:Leucyl Aminopeptidase