Magnesium Isotope Effects in Enzymatic Phosphorylation

Abstract

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 Mg(2+) ion has a magnetic isotopic nucleus 25Mg was found to be 2-3 times higher than that of enzymes in which Mg(2+) 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 Mg(2+) 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.

Reexamination of magnetic isotope and field effects on adenosine triphosphate production by creatine kinase

“…In the real-time assay, we were limited to fields of 10 mT. However, the data of Buchachenko et al (12,15) would indicate that with this field the activity of CK in the presence of 25 Mg should increase by approximately 10%. Certainly, we would expect to have seen any such increase in the slope of the assay time course.…”

“…According to BK (17), the radical-pair-based mechanism and resulting isotope effect operate only at elevated intracellular Mg concentrations (10.5-24 mM), whereas at normal physiological concentrations (<1 mM) the classical nucleophilic mechanism (23,24) is at work. No effect was found for the reverse reaction (ATP → ADP) (12).…”

“…In a series of papers by Buchachenko and Kuznetsov (BK) together with various co-workers (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22), a radical pair mechanism has been proposed for enzymatic phosphorylation of adenosine diphosphate (ADP) to adenosine triphosphate (ATP). This mechanism is influenced by the presence of a magnesium isotope with a nuclear spin.…”

“…These enzymes are mitochondrial ATP synthase (8,11,16), phosphoglycerate kinase (9, 13), pyruvate kinase (12), and creatine kinase (9,12,15,20). The effects increased linearly with the amount of 25 Mg in the isotopic mixture (13).…”

“…Furthermore, the activity of an enzyme that contains 25 Mg was strongly enhanced in the presence of the magnetic field. For instance, with creatine kinase the 2.3-fold increase seen for 25 Mg 2þ without a field rose to a fourfold increase at 80 mT (12,15). According to BK (17), the radical-pair-based mechanism and resulting isotope effect operate only at elevated intracellular Mg concentrations (10.5-24 mM), whereas at normal physiological concentrations (<1 mM) the classical nucleophilic mechanism (23,24) is at work.…”