Kinetic properties and thermal stabilities of mutant forms of mitochondrial aspartate aminotransferase.
Kinetic properties and thermal stabilities of the precursor form of mitochondrial aspartate aminotransferase, the mature form lacking 9 amino acids from the N-terminus, and forms of the mature protein in which cysteine-166 had been mutated to serine or alanine were compared with those of the mature enzyme. The precursor and the cysteine mutants showed moderately impaired catalytic properties consistent with decreased ability to undergo transition from the open to the closed conformation which is an integral part of the mechanism of action of the enzyme. The deletion mutant had a kcat only 2% of that of the mature enzyme but also much reduced Km values for both substrates. In addition it showed enhanced reactivity of cysteine-166 with 5,5'-dithiobis(2-nitrobenzoate), which is characteristic of the closed form of the enzyme, with no enhancement of reactivity in the presence of substrates. This is taken to show that the deletion mutant adopts a conformation that is significantly different from that of the mature enzyme particularly in respect of the small domain. The deletion mutant was found to be more resistant to thermal inactivation over a range of temperatures than were the other forms of the enzyme consistent with its having a more tightly packed small domain.
Centro di Studio sui Mitocondri e Metabolismo Energetico, CNR, Bari and Trani, Italy.
A
A
Azzariti
R A
RA
Vacca
S
S
Giannattasio
R S
RS
Merafina
E
E
Marra
S
S
Doonan
D000818:Animals; D001219:Aspartate Aminotransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002645:Chickens; D004795:Enzyme Stability; D006358:Hot Temperature; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology*; D009154:Mutation; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering; D011498:Protein Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D013329:Structure-Activity Relationship
D007527:Isoenzymes; D010446:Peptide Fragments; D011498:Protein Precursors; D001219:Aspartate Aminotransferases