The use of nucleotide phosphorothioate diastereomers to define the structure of metal-nucleotide bound to GTP-AMP and ATP-AMP phosphotransferases from beef-heart mitochondria.
The diastereomers of adenosine 5'-O-[1-thio]triphosphate (ATP[alpha S]) and adenosine 5'-O-[2-thio]triphosphate (ATP[beta S]) were utilized to seek unambiguous assignment of Mg2+ coordination to ATP when bound to ATP-AMP phosphotransferase from beef heart mitochondria (AK2). Similarly, the diastereomers of guanosine 5'-O-[thio]triphosphate (GTP[alpha S]) and guanosine 5'-O-[2-thio]triphosphate (GTP[beta S]) were utilized to seek unambiguous assignment of Mg2+ coordination to GTP when bound to GTP-AMP phosphotransferase from beef heart mitochondria (AK3). Furthermore the diastereomers of guanosine 5'-O-[1-thio]diphosphate (GDP-[alpha S]) have been used to assign Mg2+ coordination to GDP when bound to AK3. The ratios (V for isomer Sp)/(V for isomer Rp) obtained in the presence of Mg2+ and Cd2+ are compared to those already published for ATP-AMP phosphotransferases from pig muscle (AK1) [Kalbitzer et al. (1983) Eur. J. Biochem. 133, 221-227] and from baker's yeast (AKy) [Tomasselli and Noda (1983) Eur. J. Biochem. 132, 109-115]. In all cases, coordination of Mg2+ to the beta-phosphate via the pro-R oxygen is present, as shown by reversal of specificity for the diastereomers of ATP [beta S] or GTP [beta S] respectively on changing the metal ion. In contrast, there is no reversal of specificity for the diastereomers of ATP [alpha S] or GTP[alpha S], or for GDP[alpha S] in the case of AK3 for the reverse reaction, indicating that there is no interaction of the metal with the alpha-phosphate group. The observed stereospecificity for the alpha-thiophosphate is consistent with the assumption of an interaction of the pro-R oxygen of the alpha-phosphate group with the enzyme.
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000263:Adenylate Kinase / Q000378:metabolism*; D000345:Affinity Labels; D000818:Animals; D002104:Cadmium / Q000378:metabolism; D002417:Cattle; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000031:analogs & derivatives*; D006153:Guanosine Diphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D008274:Magnesium / Q000378:metabolism*; D008929:Mitochondria, Heart / Q000201:enzymology*; D009703:Nucleoside-Phosphate Kinase; D010770:Phosphotransferases / Q000378:metabolism*; D013237:Stereoisomerism; D013379:Substrate Specificity; D013873:Thionucleotides / Q000378:metabolism*
D000345:Affinity Labels; D013873:Thionucleotides; C017817:adenosine 5'-(1-thio)triphosphate; C034882:guanosine 5'-O-(1-thiotriphosphate); C034883:guanosine 5'-O-(2-thiotriphosphate); D002104:Cadmium; D006153:Guanosine Diphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C022570:adenosine 5'-O-(2-thiotriphosphate); C023427:guanosine 5'-O-(2-thiodiphosphate); D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D010770:Phosphotransferases; C021894:nucleoside triphosphate-adenylate kinase; D000263:Adenylate Kinase; D009703:Nucleoside-Phosphate Kinase; D008274:Magnesium