Mitochondrial adenylate kinase (AK2) from bovine heart. The complete primary structure

Abstract

1. The sequence analysis of adenylate kinase isoenzyme 2 (AK2) was completed using a gas-phase sequencer constructed in our laboratory. The enzyme contains 238 amino acid residues in the following order : The four cysteine residues of AK2 were reinvestigated. Cys-41 and Cys-233 contain free thiols, which can be carboxymethylated in the intact protein without loss of enzymic activity. Chemical and model-building studies suggest that the pair Cys-43/Cys-93 forms a disulfide in native AK2.3. The relative molecular mass of AK2, as deduced from the sequence, is 26104. Other methods, including titration of -SH groups, sedimentation equilibrium ultracentrifugation and gel filtration yielded M , values in the range from 26000 to 31 500, each value depending on the respective method of determination.4. Bovine heart AK2 contains 44 residues more than the homologous isoenzyme AK1 (myokinase). As all but one single insertions and deletions cancel, the higher M , of AK2 is due to 9 residues preceding the N terminus of AK1, a stretch of 30 residues in the middle of the molecule and 6 residues at the end.5. AK2 and AK1 are similar in their active-site geometry. In contrast, AK2 does not possess any of the three antigenic sites of AK1, which is consistent with the lack of immunological cross-reactivity between AK1 and AK2.

Substitution of a serine residue for proline-87 reduces catalytic activity and increases susceptibility to proteolysis of Escherichia coli adenylate kinase.

“…(i) Comparison of E. coli adenylate kinase with the pig muscle enzyme and other recently sequenced adenylate kinases (28,29) showed a rather high degree of homology (about 30%), most evident at the N-terminal half of the molecule. In all these enzymes Pro (conserved as Pro-96 in pig muscle adenylate kinase) is part of an invariant sequence, Gly-Phe-Xaah-Xaah-Asp-Gly-XaaA,-Pro-Arg (Xaah represents the hydrophobic residue Ile or Leu, and XaaAr represents the aromatic residue Phe or Tyr).…”

Guanylate kinase from Saccharomyces cerevisiae

“…K S K I I F -V X 7 G G P G S G K G T Q C E . pig AK1 [ZO], AK2 from bovine heart mitochondrial intermembrane space [46], AK3 from bovine heart mitochondrial matrix [47], AKyst from baker's yeast [21], AKeco from E. coli [48] and AKpdn from Paracoccus denitrifcans [6]. The bottom numbers refer to pig AK1 [20]; residue numbers of other adenylate kinases can be taken from the alignment of [49].…”

“…Since we are searching for very distant relationships and since all known small variants are closely related to each other (72 -98% identical amino acid residues [S]), they were represented by the structurally best known species, pig AK1 [20,34]. The large variants have only 30-52% identical amino acid residues [6], so that we decided to scrutinize all known sequences : AK2 from mitochondrial intermembrane space [46], AK3 from mitochondrial matrix [47], AKyst from yeast [21], AKeco from E. coli [48] and AKpdn from Paracoccus denitrijicans [6]. The overall alignment within the adenylate kinase family is now supported by more than three three-dimensional structures [4, 5, 34 -361, rendering substantial errors rather unlikely.…”

Substrate‐binding and catalytic roles of Lys194 in the C‐terminus in human adenylate kinase by site‐directed random mutagenesis

“…The AK family consists of three isoenzymes: AK1, AK2, and AK3. AK1 is localized in mammalian cytosol (5), AK2 in mitochondria intermembrane space (6,7), and AK3 in mammalian mitochondria matrix (8). The molecular weight of human AK1 (hAK1) is 21.7 kDa known to be one of the smallest phosphotransferases.…”