The complete amino acid sequence of adenylate kinase from baker's yeast

Abstract

The complete amino acid sequence of cytosolic adenylate kinase (MgATP + AMP P MgADP + ADP) from 1. Tryptic and clostripaic cleavage of the protein yielded 27 and 10 fragments, respectively. They were 2. Alignment of the clostripaic fragments was deduced from the sequence of peptides obtained by 3. Thc N-terminus is blocked by an acetyl group as shown by proton magnetic resonance. 4. Carboxypeptidase A digestion of the whole protein showed that the C-terminal sequence is -Lys-Asn, in agreement with the sequence of peptides from tryptic, clostripaic and 2-iodosobenzoic acid cleavages.5. The enzyme is a monomer of 220 amino acids with M , 24077. Comparison of the sequence of the cytosolic adenylate kinases from yeast and pig shows 25% identity with highly conserved segments in the putativc activesite region of the enzyme. After position 11 1, however, there is an insertion of 32 residues in thc yeast species, similar to the adenylate kinase and the GTP : AMP phosphotransferase from beef heart mitochondria.baker's yeast has been determined. sequenced with either a solid-phase sequencer or a gas-phase sequencer. [5] and of two isozymes from beef heart mitochondria [6, 7] have been reported. An X-ray diffraction analysis of porcine adenylate kinase at 0.3 nm in conjunction with the sequence yielded the spatial structure [8]. Whereas the active-site region of the enzyme could be assigned unambiguously by localization of substrate analogues at 0.6-nm resolution 191, the exact binding positions of the substrates are still not known. However, the observed binding sites were challenged by nuclear magnetic resonance analyses [lo]. This controversy is likely to be resolved by study of adenylate kinase from yeast (AKy), because it cocrystallizes with the potent enzyme inhibitor Ap,A as well as with the natural substrate MgATP (Noda and Tomasselli, unpublished results). These crystals are presently being analyzed by X-ray diffraction. This paper presents the complete amino acid sequence of AKy, which together with the forthcoming X-ray analysis should yield the exact substrate binding geometry, leading us a step closer to the elucidation of the reaction mechanism. MATERIALS AND METHODS MaterialsBaker's yeast (Red Star Yeast, Universal Foods Corporation, Milwaukee, WI) was kindly donated by the manufacturer. Clostripain from Clostridiurn histolyticurn, endoproteinase Lys-C from Lysohacter enzyrnogenes, pronase from Streptornyces griseus (Boehringer Mannheim), Staphylococcus aureus V 8 protease (Miles Laboratories Inc.) and trypsin pretreated with L-3-tosyl-amido-2-phenylethylchloromethylanc (Worthington) were used for proteolytic cleavages. Cyanogen bromide (Fluka) and 2-iodosobenzoic acid (Serva) were employed for chemical cleavages. Reagents and solvents for the gas-phase sequencer were from Applied Biosystems. All other chemicals were of analytical grade. Protein preparation and chemical mod(ficationsAKy, prepared and assayed as in [l 11, had a specific activity of 1833 U/mg and was homogeneous by sodium dodecyl sulfate/pol...

High‐resolution structures of adenylate kinase from yeast ligated with inhibitor Ap5A, showing the pathway of phosphoryl transfer

“…Sequence and secondary structures. Upper line, amino acid sequence (Tomasselli et al, 1986); second line, manually assigned secondary structures (E, &sheet; H, a-helix; G , 310-helix) for both structures; third line, assigned names of &strands and a-helices. &Strands of LID are named separately because this domain is not present in small variants.…”

“…Here we report two refined structures of the adenylate kinase from yeast, which is a large variant and consists of 220 amino acid residues with an M , of 24,077 (see Kinemages 1 and 2; Tomasselli et al, 1986;Magdolen et al, 1987;Proba et al, 1987). In one crystal form that has been elucidated at 1.96 A resolution, AK,,, is ligated with the two-substrate-mimicking inhibitor Ap,A and Mg2+.…”

Redundant Mitochondrial Targeting Signals in Yeast Adenylate Kinase

“…In the steady state, cytoplasmic precursor pools are usually very small (6 -8). Adenylate kinase (Adk1/Aky2p, named Aky2p hereafter (9)) deviates from this general scheme in important aspects: (i) Aky2p occurs at two subcellular locations, the cytoplasm and mitochondrial intermembrane space (IMS), and most of Aky2p is excluded from entry into mitochondria; (ii) it lacks a cleavable presequence, and at both locations Aky2p is derived from the same open reading frame and is identical with respect to primary structure and posttranslational modifications (10,11); (iii) the N-terminal peptide, which carries import information, is short (7-8 amino acids), electrically neutral, and displays a very low hydrophobic ␣-helical moment (12).…”

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

“…Thus our data are consistent with the epitope assignments in AK1. Surprisingly AK2 was found to crossreact immunologically with adenylate kinase from yeast [14], an enzyme of known sequence [28]. The comparison of the antigenic sites in mitochondria1 AK2 with those of cytosolic yeast AK is expected to answer a number of phylogenetic and clinical questions concerning the cross-reactivity of microbial and mammalian proteins [14].…”