Shift in pH-rate profile and enhanced discrimination between dicarboxylic and aromatic substrates in mitochondrial aspartate aminotransferase Y70H.
Tyr70 of chicken mitochondrial aspartate aminotransferase was replaced with a histidine residue by oligonucleotide-directed mutagenesis. Aspartate aminotransferase Y70H retained at pH 7.5 13% of the activity toward dicarboxylic amino acids, whereas the activity toward aromatic amino acids was only 0.6% of that of the wild-type enzyme, corresponding to a 22-fold increase in the ratio of the activities toward these two types of substrates. In comparison to that of the wild-type enzyme, the low-pH limb of the pH-activity profile of the mutant enzyme was shifted to higher pH values, very likely reflecting the titration curve of the newly introduced histidine residue with a pKa' of 6.3. Apparently, a positively charged residue at position 70 abolishes enzymic activity. The spectrophotometrically determined pKa' value of the internal aldimine formed between pyridoxal 5'-phosphate and Lys258 in the mutant enzyme was 6.0, similar to that in the wild-type enzyme. The rate constant of the dissociation of pyridoxamine 5'-phosphate from the mutant enzyme was increased only 3 times over that of the wild-type enzyme, in contrast to the 80-fold increase in Escherichia coli aspartate aminotransferase Y70F [Toney, M. D., & Kirsch, J. F. (1987) J. Biol. Chem. 262, 12403-12405], suggesting that His70 can replace Tyr70 in forming a hydrogen bond to the coenzyme.
Pan
P
P
Biochemisches Institut, Universität Zürich, Switzerland.
Jaussi
R
R
Gehring
H
H
Giannattasio
S
S
Christen
P
P
D000595:Amino Acid Sequence; D000818:Animals; D001219:Aspartate Aminotransferases / Q000737:chemistry / Q000378:metabolism*; D001224:Aspartic Acid / Q000378:metabolism; D002645:Chickens; D002942:Circular Dichroism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000378:metabolism; D017354:Point Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013053:Spectrophotometry; D013379:Substrate Specificity; D014364:Tryptophan / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism
D011994:Recombinant Proteins; D001224:Aspartic Acid; D014443:Tyrosine; D010649:Phenylalanine; D014364:Tryptophan; D001219:Aspartate Aminotransferases