Metal-ATP complexes

As discussed earlier, the enzymic reaction catalyzed by glutamine synthetase requires the presence of divalent metal ions. Extensive work has been conducted on the binding of Mn2+ to the enzyme isolated from E. coli (82, 109-112). Three types of sites, each with different affinities for Mn2+, exist per dodecamer n, (12 sites, 1 per subunit) of high affinity, responsible for inducing a change from a relaxed metal ion free protein to a conformationally tightened catalytically active protein n2 (12 sites) of moderate affinity, involved in active site activation via a metal-ATP complex and n3 (48 sites) of low affinity unnecessary for catalysis, but perhaps involved in overall enzyme stability. The state of adenylylation and pH value alter the metal ion specificity and affinities. 

Figure 13-2 (a) Structure of adenosine triphosphate (ATP), with ligand atoms shown in color. ib) Possible structure of a metal-ATP complex, with four bonds to ATP and two bonds to H2O ligands. 

Considering that each metal possesses its own spectra of affinity constants for different biological ligands, the values of stability constants have been used as descriptors for QSAR studies. Some examples include the log of the equilibrium constant (log K q) of a metal-ATP complex (Biesinger and Christensen 1972), the stability constant of metal-ion complexes with NH3, the stability constants of metal ion complexes with EDTA, and the stability constants of divalent metal ions with AMP (Enache et al. 2003). 

Log of the equilibrium constant (log of the metal-ATP complex Covalent index (X r)... 

Sigman et al. (1972) have reported an example of metal-ion facilitated phosphorylation of an alcohol by adenosinetriphosphate (ATP). Complexing of the metal ion, ATP, and 2-hydroxymethyl-... 

The equilibrium constant of an enzyme-catalyzed reaction can depend greatly on reaction conditions. Because most substrates, products, and effectors are ionic species, the concentration and activity of each species is usually pH-dependent. This is particularly true for nucleotide-dependent enzymes which utilize substrates having pi a values near the pH value of the reaction. For example, both ATP" and HATP may be the nucleotide substrate for a phosphotransferase, albeit with different values. Thus, the equilibrium constant with ATP may be significantly different than that of HATP . In addition, most phosphotransferases do not utilize free nucleotides as the substrate but use the metal ion complexes. Both ATP" and HATP have different stability constants for Mg +. If the buffer (or any other constituent of the reaction mixture) also binds the metal ion, the buffer (or that other constituent) can also alter the observed equilibrium constant . ... 

A metal-nucleotide complex that exhibits low rates of ligand exchange as a result of substituting higher oxidation state metal ions with ionic radii nearly equal to the naturally bound metal ion. Such compounds can be prepared with chromium(III), cobalt(III), and rhodi-um(III) in place of magnesium or calcium ion. Because these exchange-inert complexes can be resolved into their various optically active isomers, they have proven to be powerful mechanistic probes, particularly for kinases, NTPases, and nucleotidyl transferases. In the case of Cr(III) coordination complexes with the two phosphates of ATP or ADP, the second phosphate becomes chiral, and the screw sense must be specified to describe the three-dimensional configuration of atoms. 

Preliminary rate measurements should allow one to make a plot of initial velocity Vq versus [metal ion], and this should provide information on the optimal metal ion concentration. (For many MgATP -dependent enzymes, the optimum is frequently 1-3 mM uncomplexed magnesium ion.) Then, by utilizing pubhshed values for formation constants (also known as stability constants) defining metal ion-nucleotide complexation, one can readily design experiments to keep free metal ion concentration at a fixed level. To compensate properly for metal ion complexation in ATP-dependent reactions, one must chose a buffer for which a stability constant is known. For example, in 25 mM Tris-HCl (pH 7.5), the stability constant for MgATP is approximately 20,000 M Thus, one can write the following equation ... 

Chromium(III) forms stable complexes with adenosine-S -triphosphate.840,841,842 These are kinetically inert analogues of magnesium ATP complexes and may be used to study enzyme systems. The complexes prepared are chiral and may be distinguished in terms of chirality at the metal centre (198,199).843 The related complex of chromium(lll) with adenosine-5 -(l-thiodiphosphate) has been prepared the diastereoisomers were separated.844 The stereospecific synthesis of chromium(III) complexes of thiophosphates has been reported845 by the method outlined in equation (47), enabling the configuration of the thiophosphoryl centre to be determined. The availability of optically pure substrates will enable the stereospecificity of various enzyme systems to be investigated.845... 

Suzuki et al. examined the effect of various divalent cations on purified recombinant human GCH expressed in Escherichia coli to clarify the molecular mechanism of action of divalent cations on the GCH enzymatic activity [150]. They demonstrated that GCH utilizes metal-free GTP as the substrate for the enzyme reaction. Inhibition of the GCH activity by divalent cations such as Mg(II) and Zn(II) was due to a reduction in the concentration of metal-free GTP substrate by complex formation. Many nucleotidehydrolyzing enzymes such as G proteins and kinases recognize Mg-GTP or Mg-ATP complex as their substrate. In contrast with these enzymes, Suzuki et al. demonstrated that GCH activity is dependent on the concentration of Mg-free GTP [150]. 

In the case of ATP, it has been possible to observe interactions with specific phosphate groups due to formation of complexes with Cu2+, Mn2+ and Co2+. The addition of Cu2+ indicates a complex which involves only the / and y phosphates while Mn2+ seems to form complexes with ATP involving all three phosphate groups as displayed by broadening of all three P peaks. Co2+ affects the spectrum in a qualitative manner similar to Mn2+. The exemption of Cu2+ is not surprising since this metal ion is able to form additional intermediate or metal-ring complexes. These... 

The true substrates for many enzymes which utilize nucleotides as substrates are metal-nucleotide complexes, often Mg(II)-nucleotides such as Mg-ATP. Numerous... 

A central question in phosphotransferases and nucleotidyltransferases is the structure of the metal-nucleotide complex which is the true substrate for the enzyme. It is unlikely that all of the possible Mg-ATP complexes could serve as substrates for a given enzyme, but until recently there has been no way to determine which isomer is active. The difficulty is the coordination exchange equilibrium, which is rapidly set up and dynamically maintained in solutions of Mg-ATP. To avoid this problem, metal-nucleotide complexes have been synthesized using coordination exchange-inert metals such as Cr(III) and Co(IIl) in place of Mg(II) [7,60], The resulting complexes are structurally stable and can be separated by chromatographic methods into their coordination isomers and stereoisomers. The isomers can then be investigated as substrates or inhibitors of specific enzymes. 

The simplest use of manganese is as part of a metal cofactor such as manganese(II) adenosine triphosphate, [Mn(II)ATP2-]. The metal complex is usually found as the bidentate [Mn(II)0,7-ATP2 ] (3, 4) as illustrated in Figure 1. Enzyme specificity for Mg(II), Mn(II), or Ca(II) ATP complexes is dependent on a variety of factors however, once selected, each metal apparently functions in an analogous manner. The metal ion serves two main purposes. First, based on the coordination geometry... 

As found with H2P20 and H2P30 hydrolysis of ATP and ADP can be accelerated by [Co(OH)(OH2)(N)4]2+ species570,571 and by the hydrolysis products of [Co(Cl)3(dien)].572 The (N)4 = (tn)2 complex shows no acceleration for a 1 1 ratio, but the 2 1 metal ATP mixture (0.1-0.01 mol dm 3 ATP) shows 105 enhancement at pH 7. Scheme 63 summarizes these findings. At high pH hydrolysis of the Coin complex predominates. [Co(cyclen)(H20)2]3+ also catalyzes the hydrolysis of [Co(NH3)4(ATP)] at pH 8-9, and (168) has been claimed as the reactive species.581 In all these studies however no intermediates have been positively identified, and the immediate products, coordinated or otherwise, also lack definition. 

The true substrate of FqFj is MgATP, rather than the free nucleotide. There is a strict stereochemical requirement for the structure of metal-nucleotide complexes in the phosphorylation reaction [161]. In the case of myosin ATPase, for example, the Sp diastereoisomer (A in Fig. 5.9) of ATP)8S is more rapidly hydrolyzed than its Rp diastereoisomer (B in Fig. 5.9) with Mg with an Sp/Rp ratio of > 3000, while with... 

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