| Abstract
| - Pigeon liver malic enzyme has an N-terminal amino acid sequence of Met-Lys-Lys-Gly-Tyr-Glu-Val-Leu-Arg-. Our previous results indicated that the N-terminus of the enzyme is located at or near the enzyme's active center involved in Mn(II)-L-malate binding and is also near to the subunits' interface. In the present study, the conformational stability of the various deletion (delta) and substitution mutants at Lys2/Lys3 of the enzyme was investigated with chemical and thermal sensitivities. The lysine residue at position 2 or 3 seems to be crucial for the correct active site conformation, probably through an ion-pairing with Glu6. Deletion at Lys2 or Lys3, delta(K2/K3), and the double mutant K(2,3)E were much less stable than the wild-type enzyme towards chemical denaturation. Kinetic analysis of the thermal inactivation at 58 degrees C of the recombinant enzymes indicated that mutation at position 3 to alanine (K3A) endows the protein with extra stability compared with the wild-type enzyme. K3A is also stable towards chemical denaturation. The concentration of urea that causes half unfolding, [urea]0.5, for K3A is 3.25 M compared with 2.54 M for the wild-type enzyme. The K3A mutant of malic enzyme might therefore have potential practical applications.
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