Complexes of yeast adenylate kinase and nucleotides investigated by 1H NMR.
The role of one of the histidine residues present in many adenylate kinases (H36 in the porcine cytosolic enzyme) is highly disputed. We thus studied the yeast enzyme (AKye) containing this His residue. AKye is highly homologous to the Escherichia coli enzyme (AKec), a protein that is already well characterized by NMR [Vetter et al. (1990) Biochemistry 29, 7459-7467] and does not contain the His residue in question. In addition, discrepancies between solution structural and X-ray crystallographic studies on the location of the nucleotide binding sites of adenylate kinases are clarified. One- and two-dimensional nuclear magnetic resonance (NMR) spectroscopy was used to investigate AKye and its complex with the bisubstrate analogue P1,P5-bis(5'-adenosyl)pentaphosphate (AP5A). The well-resolved spectra of AKye allowed identification of nearly all detectable resonances originating from aromatic side chain protons (12 out of 15 spin systems). From these studies, all aromatic residues of AKec involved in the binding of ATP.Mg2+ have functional analogues in AKye. The AMP site seems to make no contacts to aromatic side chains, neither in the AKye.AP5A.Mg2+ nor in the AKec.AP5A.Mg2+ complexes, so that it is presently not possible to localize this binding site by NMR. The ATP site of AKye is located near residues W210 and H143 in a position similar to the ATP site of the E. coli enzyme. In combination with the recent X-ray results on the AP5A complexes AKye and AKec and the GMP complex of guanylate kinase [Stehle, T., & Schultz, G. E. (1990) J. Mol. Biol. 221, 255-269], the latter one leading to the definition of the monophosphate site, the problem of the location of the nucleotide sites can be considered to be solved in a way contradicting earlier work [for a review, see Mildvan, A. S. (1989) FASEB J. 3, 1705-1714] and denying the His residue homologous to H36 in porcine adenylate kinase a direct role in substrate binding.
Vetter
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Department of Biophysics, Max Planck Institute for Medical Research, Heidelberg, FRG.
Konrad
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Rösch
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D000263:Adenylate Kinase / Q000378:metabolism*; D000818:Animals; D015226:Dinucleoside Phosphates / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D008274:Magnesium / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D009711:Nucleotides / Q000378:metabolism*; D011487:Protein Conformation; D011522:Protons; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013552:Swine
D015226:Dinucleoside Phosphates; D009711:Nucleotides; D011522:Protons; C012275:P(1),P(5)-di(adenosine-5'-)pentaphosphate; D000263:Adenylate Kinase; D008274:Magnesium