Mitochondrial adenylate kinase (AK2) from bovine heart. Homology with the cytosolic isoenzyme in the catalytic region

Abstract

The adenylate kinase isoenzyme located in the intermembrane space of mitochondria, AK2, is a monomeric protein of MW 30,000 which catalyzes the reaction ATP + AMP .dblarw. 2ADP. The protein was reduced and S-carboxymethylated with iodo[14C2]acetate. Using a Laursen sequenator, the N-terminal sequence of S-carboxymethylated AK2 was determined as Ala-Pro-Asn-; in some batches of the isolated protein the N-terminal dipeptide portion was missing. The C-terminus of AK2 was Met. Cleavage was CNBr yielded 8 fragments which could be isolated in 1 step using high-performance size-exclusion chromatography. They ranged in size over 4-88 amino acid residues, the total being .apprx. 270 residues. All CNBr fragments were overlapped with Met-containing tryptic peptides of AK2. The N-terminal 111 residues of AK2 were sequenced. Except for an N-terminal extension of 9 residues, this segment of AK2 could be aligned with the sequence 1-104 of cytosolic AK1. Allowing for 2 deletions in AK2, 43 of the 102 aligned residues are identical. Since this section contains the catalytic residues such as His-36 and Asp-93, AK1 can apparently serve as a 3-dimensional model of AK2 in mechanistic and drug-designing studies. Preliminary sequence results on AK2 beyond position 104 show that AK2 here contains a wing of .apprx. 50 residues which had no counterpart in AK1. The chain folds of the adenylate kinase isoenzymes are similar again for a position corresponding to residue 115 of AK1 onwards. The additional structural motifs of AK2 are probably related to the location of this isoenzyme in the mitochondrion.