Structural and functional role of the amino-terminal region of porcine cytosolic aspartate aminotransferase. Catalytic and structural properties of enzyme derivatives truncated on the amino-terminal side.
In porcine cytosolic aspartate aminotransferase, a dimeric enzyme, the amino-terminal region anchoring onto the neighboring subunit is linked to the adjoining floppy peptide segment (residues 12-47), an integral part of the small domain whose facile movement upon substrate binding is a striking "induced fit" feature of this enzyme. To assess the contribution by the amino-terminal region to small domain movement and protein stability, a series of enzyme derivatives truncated on the amino-terminal side (residues 1-9) was prepared by using oligonucleotide-directed in vitro mutagenesis. Deletion of residues 1-3 showed no effect on catalytic activity and heat stability. Del 1-5 mutant enzyme with an extra methionine at position 5 showed only 43% of the kappa cat value (in the overall transamination) of the wild-type enzyme. Further deletion up to residue 9 resulted in a slight decrease in kappa cat values. Del 1-9 mutant enzyme still retained a kappa cat value of 33% that of wild-type enzyme. Km values for aspartate and 2-oxoglutarate increased sharply upon deletion of residues 1-9. Accordingly, Del 1-9 mutant enzyme showed a striking decrease in the kappa cat/Km value, to only 2% of that for the wild-type enzyme. Deletion of amino-terminal residues 1-9 resulted also in a large decrease in thermostability and in an enhanced susceptibility to limited proteolysis by protease 401, which is known to cleave at Leu20 of the wild-type enzyme. These findings indicate that an increase in the conformational freedom of the floppy segment (residues 12-47) would occur upon the loss of most of the anchorage region, thereby presenting an entropic barrier to conformational changes that facilitate substrate binding with high affinity.
Fukumoto
Y
Y
Department of Urology, Kumamoto University Medical School, Japan.
Tanase
S
S
Nagashima
F
F
Ueda
S
S
Ikegami
K
K
Morino
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D001219:Aspartate Aminotransferases / Q000378:metabolism*; D001483:Base Sequence; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D006358:Hot Temperature; D007700:Kinetics; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D011487:Protein Conformation; D011489:Protein Denaturation; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013552:Swine
D001219:Aspartate Aminotransferases