CONFORMATIONAL STABILITY OF THE N-TERMINAL AMINO-ACID-RESIDUES OF MUTATED RECOMBINANT PIGEON LIVER MALIC ENZYMES

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 activ e center involved in Mn(II)-L-malate binding and is also near to the s ubunits' 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 sensitivitie s. The lysine residue at position 2 or 3 seems to be crucial for the c orrect 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 degr ees C of the recombinant enzymes indicated that mutation at position 3 to alanine (K3A) endows the protein with extra stability compared wit h the wild-type enzyme. K3A is also stable towards chemical denaturati on. 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 ap plications.