The amino acid sequence of GTP:AMP phosphotransferase from beef‐heart mitochondria

Abstract

The amino acid sequence of GTP :AMP phosphotransferase (AK3) from beef-heart mitochondria has been determined, except for one segment of about 33 residues in the middle of the polypeptide chain. The established sequence has been unambiguously aligned to the sequence of cytosolic ATP :AMP phosphotransferase (AK1) from pig muscle, allowing for six insertions and deletions. With 30 % of all aligned residues being identical, the homology between AK3 and AKI is well established. As derived from the known three-dimensional structure of AKI, the missing segment is localized at a small surface area of the molecule, far apart from the active center. The pattern of conserved residues demonstrates that earlier views on substrate binding have to be modified. The observation of three different consecutive N-termini indicates enzyme processing.The enzyme GTP: AMP phosphotransferase (AK3, Mr = 26000) catalyzes the reaction MgGTP + AMP + MgGDP + ADP. The enzyme AK3 is located in the inner matrix of the mitochondria [I -31 and is involved in the reaction sequence of substrate level phosphorylation [l]. Purification procedures, physical, chemical, kinetic and inhibition studies of AK3 from beef-heart mitochondria [2 -41 have emphasized similarities with the adenylate kinases from mammalian cytosol [3] and mitochondria [5], AKI and AK2 respectively, as well as from yeast cytosol [6]. All these enzymes are water-soluble, globular, low-M, proteins relatively stable in a wide range of p H and are purified by using similar purification protocols.The catalytic centers of adenylate kinases carry two binding sites for the nucleotides. This is borne out by the strong binding of adenosine(5')pentaphospho(5')adenosine by AK1 [7] and AK2 [S] and guanosine(5')pentaphospho(5')adenosine by AK3 [3]. Moreover, kinetic, spectrophotometric and equilibrium dialysis binding studies [9] (and citations therein) demonstrate two binding sites. One site, the AMP site, of AKI, AK2 and AK3 is very specific for AMP. Rigorous specificity does not apply to the other site. The GTP-binding site of AK3 can accommodate both guanosine and inosine nucleotides but does not accommodate ATP, CTP or UTP [4]; the ATP-binding site of AK1 and AK2 can accommodate GTP, CTP, ITP and UTP 161.The amino acid sequence and the three-dimensional structure of AK1 from pig muscle have been elucidated [lo-121. Sequences of other species have been determined completely [13] or partially [14, 151. The active-center region of AKI was established by binding studies of substrate analogs in crystals [16]. The enzyme molecule is to some extent deformed in these crystals as evidenced by the break up of crystals on binding substrates. Further X-ray analyses of other crystal forms and/or of other adenylate kinases from different species are necessary for elucidating the catalytic mechanism. The analysis of AK3 crystals is in progress [17]. In this paper we report most of the amino acid sequence of AK3 and its comparison with the sequence of AK1 and modify earlier views on substrate binding pos...

The uncoupling protein from brown fat mitochondria is related to the mitochondrial ADP/ATP carrier. Analysis of sequence homologies and of folding of the protein in the membrane.

“…In the sequence of the AAC, two insertions of one amino acid each had to be assumed between residues 202/203 and 205/206. Quantification of the sequence similarities of UCP and AAC With this alignment the similarity between the sequences was evaluated according to Wieland et al (1984). Fig.…”

“…4. Plot of local similarities between the AAC and the UCP according to Wieland et al (1984). The alignment of the sequences was performed by inserting necessary residues by hand.…”

Three-dimensional structure of the complex between the mitochondrial matrix adenylate kinase and its substrate AMP

“…The enzyme is essential for the biosynthesis of ADP from AMP. The polypeptide chain consists of 225 amino acid residues with Mr = 25 469 and belongs to the group of large variants within the adenylate kinase family (Wieland et al, 1984;Tomasselli et al, 1986).…”

“…The enzyme is essential for the biosynthesis of ADP from AMP. The polypeptide chain consists of 225 amino acid residues with M , = 25469 and belongs to the group of large variants within the adenylate kinase family (Wieland et al, 1984;Tomasselli et al, 1986). The overall sequence homology to other adenylate kinases is rather low: 37% identical amino acid residues with the large variant AKyst and 24% with the small variant AKlpig.…”

Metabolic homeostasis is maintained in myocardial hibernation by adaptive changes in the transcriptome and proteome

“…Interestingly, a chain of adenylate kinase-catalyzed phosphotranfer reactions has been implicated in the communication of mitochondria-generated signals to K(ATP) channels [20]. Mitochondrial adenylate kinase 4, identified in this study, contributes to the phosphorylation of AMP, but can only use GTP or ITP as a substrate [36]. It has recently been demonstrated to interact with the mitochondrial inner membrane protein adenine nucleotide translocase, which might be important for its protective benefits under stress conditions [37].…”