FUNCTIONAL ROLES OF THE N-TERMINAL AMINO-ACID-RESIDUES IN THE MN(II)-L-MALATE BINDING AND SUBUNIT INTERACTIONS OF PIGEON LIVER MALIC ENZYME

Pigeon liver malic enzyme has an N-terminal amino acid sequence of Met -Lys-Lys-Gly-Tyr-Glu-. In this work, various mutants of the enzyme wit h individual or combinational deletion (Delta) or substitution at thes e amino acids were constructed and functionally expressed in Escherich ia coli cells, A major protein band corresponding to an M-r of similar to 65 000 was observed for all recombinant enzymes in sodium dodecyl sulfate polyacrylamide gel electrophoresis, However, when examining by polyacrylamide gel electrophoresis under native conditions, the recom binant enzymes were found to possess a tetrameric structure with M-r s imilar to 260 000 or a mixture of tetramers and dimers with the except ion of Delta(K2K3G4) and Delta(1-16) mutants, which existed exclusivel y as dimers at the protein concentration we employed, K3A and K3E also dissociated substantially, K(2,3)A was a tetramer but K(2,3)E essenti ally existed as dimers, All tetramers and dimers were enzymatically ac tive in the gels, All mutants displayed a similar apparent K-m value f or NADP(+), The apparent K-m for L-malate and Mn(II), on the other han d, was increased by 4-27-fold for the Delta(K2/K3) and the Delta(1-16) mutants, The small binding affinity of Delta(K2/K3) with Mn(II)-L-mal ate was specific. With additional deletion at positions 3 and/or 4, th e Delta(K2K3), Delta(K2G4/K3G4) or Delta(K2K3G4) mutants exhibited sim ilar kinetic properties for the wild type, The lysine residues at the positions 2 or 3 seem to be crucial for the correct active site confor mation, The results indicate that the N-terminus of malic enzyme is lo cated at the Mn(II)-L-malate binding domain of the active center and i s also near the subunit's interface, These results were interpreted wi th our asymmetric double-dimer model for the enzyme in which the N-ter minus was involved in the head-to-tail monomer-monomer interactions bu t not the dimer-dimer interactions.