Functional roles of valine 37 and glycine 38 in the mobile loop of porcine cytosolic aspartate aminotransferase.
The functional roles of Val37 and Gly38 in porcine cytosolic aspartate aminotransferase have been studied in the site-directed mutants V37A, G38A, and G38S where the size and hydrophobic character of these residues has been altered. Previous x-ray studies have shown that Val37 and Gly38, which are part of a flexible loop, interact directly with bound substrate. From x-ray and solution experiments we find that the V37A, G38A, and G38S mutations do not cause significant perturbations to the unliganded enzyme. Replacing Val37 with a less bulky alanine residue does not affect the maximal catalytic rate (kcat), but it does increase significantly the Michaelis constants for substrates in the overall transamination reaction between aspartate and 2-oxoglutarate. On the other hand, replacing Gly38 with alanine or serine results in striking decreases in kcat to 5 and 0.6%, respectively, of the value observed for the wild-type enzyme, as well as in considerable increases in Km values. Consequently, the catalytic competence, kcat/Km, decreases by 3 orders of magnitude for G38A and by 4 orders of magnitude for G38S. Single turnover reactions of G38A and G38S with four individual substrates (aspartate, glutamate, oxalacetate, and 2-oxoglutarate) are characterized by kinetic parameters that are largely consistent with those of the overall reaction. In addition, the mutations at position 38 impair more seriously the catalytic competence of the enzyme toward C5-substrates than toward C4-substrates. We conclude that Gly38 is probably required for proper function of the enzyme because it permits a high level of flexibility for the 36-39 peptide, which in turn allows the essential substrate-induced movement of the small domain.
Pan
Q W
QW
Department of Biochemistry, Kumamoto University School of Medicine, Japan.
Tanase
S
S
Fukumoto
Y
Y
Nagashima
F
F
Rhee
S
S
Rogers
P H
PH
Arnone
A
A
Morino
Y
Y
D000588:Amines / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D001219:Aspartate Aminotransferases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D002942:Circular Dichroism; D003600:Cytosol / Q000201:enzymology; D017931:DNA Primers; D004795:Enzyme Stability; D005998:Glycine / Q000378:metabolism*; D006358:Hot Temperature; D008969:Molecular Sequence Data; D013379:Substrate Specificity; D013552:Swine; D014633:Valine / Q000378:metabolism*; D014961:X-Ray Diffraction
D000588:Amines; D017931:DNA Primers; D001219:Aspartate Aminotransferases; D014633:Valine; D005998:Glycine