Significance of the conserved Tyr352 and Asp380 residues in the catalytic activity of Bacillus stearothermophilus aminopeptidase II as evaluated by site-directed mutagenesis.
The importance of the conserved Tyr352 and Asp380 residues of Bacillus stearothermophilus aminopeptidase II (AP-II) was investigated by site-directed mutagenesis. The wild-type and mutant enzymes were expressed in recombinant Escherichia coli M15 cells and the 45-kD proteins were purified from the cell-free extracts by Ni(2+)-NTA resin. The specific activity for Tyr352 and Asp380 replacements was decreased by more than 3.5-fold. Detailed analysis of the kinetic consequences in the mutant proteins revealed that the K (m) values were increased 1.9- to 2.6-fold with respect to wild-type enzyme. Catalytic efficiencies (k (cat)/K (m)) of mutant proteins were between 3.5- and 31-fold lower than the corresponding value of the wild-type enzyme. Tryptophan emission fluorescence and circular dichroism spectra were nearly identical for wild-type and mutant enzymes. These results indicate that residues Tyr352 and Asp380 are essential for the proper function of AP-II.
Lin
Long-Liu
LL
Department of Applied Chemistry, National Chiayi University, 300 University Road, Chiayi 60083, Taiwan. llin@mail.ncyu.edu.tw
Chen
Yi-Pu
YP
Yang
Jia-Ci
JC
Hua
Yu-Wen
YW
Wang
Wen-Ching
WC
Kuo
Lih-Ying
LY
D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001224:Aspartic Acid / Q000502:physiology*; D002384:Catalysis; D002942:Circular Dichroism; D001411:Geobacillus stearothermophilus / Q000201:enzymology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011489:Protein Denaturation; D016415:Sequence Alignment; D014443:Tyrosine / Q000502:physiology*
D001224:Aspartic Acid; D014443:Tyrosine; D000626:Aminopeptidases