KINETIC-PROPERTIES AND THERMAL STABILITIES OF MUTANT FORMS OF MITOCHONDRIAL ASPARTATE-AMINOTRANSFERASE

Kinetic properties and thermal stabilities of the precursor form of mi tochondrial aspartate aminotransferase, the mature form lacking 9 amin o 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 sho wed moderately impaired catalytic properties consistent with decreased ability to undergo transition from the open to the closed conformatio n which is an integral part of the mechanism of action of the enzyme. The deletion mutant had a k(cat) only 2% of that of the mature enzyme but also much reduced K-m values for both substrates. In addition it s howed enhanced reactivity of cysteine-166 with 5,5'-dithiobis(2-nitrob enzoate), which is characteristic of the closed form of the enzyme, wi th 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 w ere the other forms of the enzyme consistent with its having a more ti ghtly packed small domain. (C) 1998 Elsevier Science B,V, All rights r eserved.