Structural Investigations of the Mg · ATP Complex at the Active Site of Porcine Adenylate Kinase Using Phosphorothioate Analogs and Electron Paramagnetic Resonance of Mn(II) with Chiral ¹⁷)‐Labelled ATP Analogs

Abstract

The interaction between ATP, divalent metal ions and the active site of adenylate kinase from muscle has been studied by two different methods. No reversal of the relative reaction rates of the two diastereomers of adenosine 5'-([a-thioltriphosphate) was observed on changing the metal ion from Mg2+ to Cd2+, suggesting that the a-phosphate group is not coordinated to the divalent metal ion in the enzyme . metal . ATP complex. This interpretation is confirmed by the lack of influence of 170 incorporated into the a-phosphate group of ATP on the electron paramagnetic resonance (EPR) spectrum of Mn2+ at the active site of the enzyme. The specificity for the Sp diastereomer thus appears to be due to an interaction of the enzyme active site with the pro-R oxygen of the a-phosphate group. In contrast, the specificity of adenylate kinase for the diastereomers of adenosine S'([p-thioltriphosphate) is reversed from S, to R, on replacing Mgz+ by Cd2+, indicating an interaction of the pro-R oxygen of the /&phosphate group of ATP with the metal ion in the enzyme . metal . ATP complex. In agreement with this, ATP which is regio-specifically labelled with 170 on the /I-phosphate group causes a linebroadening of the EPR spectrum of Mn2+ at the active site, demonstrating coordination of at least one of the P-phosphate oxygens with the metal. Using stereospecifically labelled [/7-'70]ATPs, it was found that (Rp)--170]ATP caused a line broadening in the Mn2+ EPR spectrum similar to that seen with regio-labelled [P-170]ATP, further indicating that the pro-R oxygen of the p-phosphate group of ATP is coordinated to the metal ion at the active site of adenylate kinase. These results lead to a value of 0.22-0.25 mT for the superhyperfine coupling constant of a single 170-Mn2+ interaction in a manganese-ATP-enzyme complex. ATP labelled with I7O on the ?-phosphate group has no effect on the spectrum of MnZ+ at the active site, leading to the conclusion that Mg . ATP exists predominantly as a /I-monodentate complex at the active site of adenylate kinase. From the effect of H2I7O on the EPR spectrum of Mn2+ in the Mn . ATP complex at the active site it appears most likely that three, four or fivc water molecules are bound to the metal ion.Three techniques developed in the last decade allow information to be obtained concerning the structures of metal complexes of nucleotides at enzyme active sites. All three of these methods can be used to determine which phosphate groups of the nucleotide are coordinated to the metal ion, but each approach has distinct advantages and disadvantages.The use of diastereomcrs of thiophosphate analogs of nucleotides can provide evidence not only on the site of metal ion coordination, but also on the stereochemistry at the phosphate group concerned, i.e. it can indicate which of the two diastereotopic oxygens of a non-terminal phosphate group is involved in the interaction [1,2]. Similar information can be obtained using substition-inert Co(lI1) and Cr(II1) complexes of nucleotides as stable analogs of the rapid...

Mechanism of adenylate kinase. The essential lysine helps to orient the phosphates and the active site residues to proper conformations

“…nuclei are sensitive to bond lengths and bond angles (Gorenstein,1981), the pattern of changes upon binding of Mg2+ is best interpreted to suggest that peaks b and c correspond to the sites of coordination, (ii) It has been wellestablished that Mg2+ does not bind to the substrate at the AMP site (AMP or ADP) in the resting state (Nageswara Rao et al, 1978). (iii) The results of stereochemical studies have suggested that the mode of coordination of MgATP as a substrate of AK is /?,y-bidentate (Dunaway-Mariano & Cleland, 1980; Tomasselli & Noda,1983; Kalbitzer et al, 1983), even though the coordination to Py has not been demonstrated spectroscopically (Kalbitzer et al, 1983). (iv)…”

Investigations of kinase substrate specificity with aqua rhodium(III) complexes of adenosine 5'-triphosphate

“…The structure of MgATP bound in the active site of myokinase has also been examined using several techniques other than the exchange-inert metal-ATP technique. Specifically, on the basis of 31P spin-relaxation measurements, Ray et al (1988) concluded that the structure of MgATP bound in the myokinase active site is /3,7-bidentate, and the results obtained from Mg(II)/Cd(II) ATP/3S/ATPaS studies (Tomasselli & Noda, 1981Tomasselli et al, 1984) and Mn-170-ATP EPR studies (Kalbitzer et al, 1983) indicate that the configuration at the /3-P is . Thus, the results from each of these studies provide evidence for /3,7-bidentate Mg(H20)4ATP as the myokinase substrate.…”

Phosphorus-31 NMR studies of the structure of cation-nucleotide complexes bound to porcine muscle adenylate kinase

“…Furthermore, the results of both Mn(II) EPR (Kalbitzer et al, 1983) and metal effects on nucleotide phosphorothioate stereoisomer specificity (Tomasselli & Noda, 1983;Tomasselli et al, 1984) agree that -P (ATP or GDP) is not coordinated to metal in enzymebound complexes. Indeed, the results of Mn(II) EPR (Kalbitzer et al, 1983) suggested the possibility of /3-monodentate coordination for enzyme-bound MnATP, which is not incompatible with the results of the phosphorothioate work but does present some problems with understanding the results on the exchange-inert CrATP complexes. Thus, the nature of the chelation of the obligatory divalent cation to enzyme-bound ATP (or acceptor ADP) is not clearly resolved.…”

“…Adenylate kinase is specific for the screw-sense /3,7-bidentate, exchange-inert CrATP complex whereas 3-P-glycerate kinase (Jaffe et al, 1982), creatine kinase, and arginine kinase are virtually inactive with all exchange-inert CrATP complexes (Dunaway- Mariano & Cleland, 1980). Furthermore, the results of both Mn(II) EPR (Kalbitzer et al, 1983) and metal effects on nucleotide phosphorothioate stereoisomer specificity (Tomasselli & Noda, 1983;Tomasselli et al, 1984) agree that -P (ATP or GDP) is not coordinated to metal in enzymebound complexes. Indeed, the results of Mn(II) EPR (Kalbitzer et al, 1983) suggested the possibility of /3-monodentate coordination for enzyme-bound MnATP, which is not incompatible with the results of the phosphorothioate work but does present some problems with understanding the results on the exchange-inert CrATP complexes.…”