2548
J. Phys. Chem. B 2008, 112, 2548-2556
Magnesium Isotope Effects in Enzymatic Phosphorylation Anatoly L. Buchachenko,*,† Dmitry A. Kouznetsov,† Natalia N. Breslavskaya,‡ and Marina A. Orlova§ N. N. SemenoV Institute of Chemical Physics, N. S. KurnakoV Institute of General and Inorganic Chemistry, Russian Academy of Sciences, 119991 Moscow, Russia, and M. V. LomonosoV Moscow State UniVersity, Chemical Department, 119992 Moscow, Russia ReceiVed: NoVember 19, 2007; In Final Form: December 10, 2007
Recent discovery of magnesium isotope effect in the rate of enzymatic synthesis of adenosine triphosphate (ATP) offers a new insight into the mechanochemistry of enzymes as the molecular machines. The activity of phosphorylating enzymes (ATP-synthase, phosphocreatine, and phosphoglycerate kinases) in which Mg2+ ion has a magnetic isotopic nucleus 25Mg was found to be 2-3 times higher than that of enzymes in which Mg2+ ion has spinless, nonmagnetic isotopic nuclei 24Mg or 26Mg. This isotope effect demonstrates unambiguously that the ATP synthesis is a spin-dependent ion-radical process. The reaction schemes, suggested to explain the effect, imply a reversible electron transfer from the terminal phosphate anion of ADP to Mg2+ ion as a first step, generating ion-radical pair with singlet and triplet spin states. The yields of ATP along the singlet and triplet channels are controlled by hyperfine coupling of unpaired electron in 25Mg+ ion with magnetic nucleus 25Mg. There is no difference in the ATP yield for enzymes with 24Mg and 26Mg; it gives evidence that in this reaction magnetic isotope effect (MIE) operates rather than classical, mass-dependent one. Similar effects have been also found for the pyruvate kinase. Magnetic field dependence of enzymatic phosphorylation is in agreement with suggested ion-radical mechanism.
1. Introduction
TABLE 1: Magnesium Isotopes
Phosphorylating enzymes are known to be perfectly arranged molecular devices generating energy carrier P-O chemical bond in adenosine triphosphate (ATP) and triphosphates of other nucleotides. The great progress in the knowledge of structure and understanding of molecular dynamics and mechanical functioning of ATP synthesizing enzymes are now attained.1-5 Generally accepted and quite evident mechanism of ATP synthesis implies a nucleophilic addition of inorganic phosphate (in ATP synthase) or phosphate group of phosphorylating substrates (in kinases) to adenosine diphosphate (ADP). This mechanism seems to satisfactorily explain how molecular motion in these enzymes results in chemical reaction of P-O bond formation, and how mechanical energy accumulated in enzyme and dispersed over numerous unequilibrium conformations of its macromolecule transforms into the energy of chemical bond by mechanical compression of reagents in the catalytic site. Nevertheless, despite the great progress in our knowledge of structure and understanding of molecular dynamics and functioning of ATP synthesizing enzymes, a chemical mechanism of phosphorylation remains enigmatic.1 A remarkable and intriguing property of the phosphorylating enzymes is that they are magnesium dependent. The main function of Mg2+ ion was traditionally thought to coordinate reagents, to keep them along the reaction pathway, and perhaps to slightly modify their * To whom correspondence should be addressed. E-mail:
[email protected]. † N. N. Semenov Institute of Chemical Physics, Russian Academy of Sciences. ‡ N. S. Kurnakov Institute of General and Inorganic Chemistry, Russian Academy of Sciences. § Moscow State University.
isotope 24
Mg 25 Mg 26 Mg
Spin
magnetic moment (µB)
natural abundance, %
0 5/2 0
0 0.85 0
79 10 11
chemical reactivity by complexation accompanied by redistribution of charges in a complex. The Mg2+ ion was always considered as an assistant, and it was never assumed that the ion participates directly in the phosphorylation reaction as a reagent. A new insight into the chemical mechanism of the phosphorylation stems from the recently discovered unusual and surprisingly large isotope effects of magnesium on the phosphorylating activity of enzymes.6-9 The goal of the presented paper is to summarize these results and discuss new ideas that follow from the observation of magnesium isotope effects in enzymatic phosphorylation. 2. Materials And Experimental Techniques So far as magnesium isotope effect in phosphorylation is an unexpected and unusual phenomenon, it is worthy to shortly describe materials and technologies used in isotopic biochemical experiments. Isotope-containing MgCl2 samples were obtained using treatment of magnesium oxides 24MgO, 25MgO, 26MgO, and *MgO with analytically pure HCl (*Mg means magnesium with natural abundance of the three isotopes; see Table 1). Myocardial mitochondria, many enzymes of which are Mg2+dependent, were tested in vitro.7 Mitochondrial pellets were suspended in a homogenization buffer and then divided into two portions: the first one was used for further estimation of the total ATP production in isolated mitochondria; in the second one the mitochondrial creatine kinase activity was measured as the amount of 32P-ATP formed at the incubation with
10.1021/jp710989d CCC: $40.75 © 2008 American Chemical Society Published on Web 02/05/2008
Magnesium Isotope Effects in Enzymatic Phosphorylation
J. Phys. Chem. B, Vol. 112, No. 8, 2008 2549
TABLE 2: Elemental Composition (in Mass %) of Magnesium Oxides element Mg Mn Fe Cu Ni Zn Pb Cr Ti K Sr Ba Ca
24
MgO (24
99.6 Mg) 6 × 10-3 3 × 10-3
