Phosphagens are phosphorylated guanidino compounds that are linked to energy state and ATP hydrolysis by corresponding phosphagen kinase reactions: phosphagen + MgADP + H(+) <--> guanidine acceptor + MgATP. Eight different phosphagens (and corresponding phosphagen kinases) are found in the animal kingdom distributed along distinct phylogenetic lines. By far, the creatine phosphate/creatine kinase (CP/CK) system, which is found in the vertebrates and is widely distributed throughout the lower chordates and invertebrates, is the most extensively studied phosphagen system. Phosphagen kinase reactions function in temporal ATP buffering, in regulating inorganic phosphate (Pi) levels, which impacts glycogenolysis and proton buffering, and in intracellular energy transport. Phosphagen kinase reactions show differences in thermodynamic poise, and the phosphagens themselves differ in terms of certain physical properties including intrinsic diffusivity. This review evaluates the distribution of phosphagen systems and tissue-specific expression of certain phosphagens in an evolutionary and functional context. The role of phosphagens in regulation of intracellular Pi levels likely evolved early. Thermodynamic poise of the phosphagen kinase reaction profoundly impacts this capacity. Furthermore, it is hypothesized that the capacity for intracellular targeting of CK evolved early as a means of facilitating energy transport in highly polarized cells and was subsequently exploited for temporal ATP buffering and dynamic roles in metabolic regulation in cells displaying high and variable rates of aerobic energy production.

Arginine kinase belongs to the family of enzymes, including creatine kinase, that catalyze the buffering of ATP in cells with f luctuating energy requirements and that has been a paradigm for classical enzymological studies. The 1.86-Å resolution structure of its transition-state analog complex, reported here, reveals its active site and offers direct evidence for the importance of precise substrate alignment in the catalysis of bimolecular reactions, in contrast to the unimolecular reactions studied previously. In the transitionstate analog complex studied here, a nitrate mimics the planar ␥-phosphoryl during associative in-line transfer between ATP and arginine. The active site is unperturbed, and the reactants are not constrained covalently as in a bisubstrate complex, so it is possible to measure how precisely they are pre-aligned by the enzyme. Alignment is exquisite. Entropic effects may contribute to catalysis, but the lone-pair orbitals are also aligned close enough to their optimal trajectories for orbital steering to be a factor during nucleophilic attack. The structure suggests that polarization, strain toward the transition state, and acid-base catalysis also contribute, but, in contrast to unimolecular enzyme reactions, their role appears to be secondary to substrate alignment in this bimolecular reaction.

Arginine kinase (AK) is a member of the guanidino kinase family that plays an important role in buffering ATP concentration in cells with high and fluctuating energy demands. The AK specifically catalyzes the reversible phosphoryl transfer between ATP and arginine. We have determined the crystal structure of AK from the horseshoe crab (Limulus polyphemus) in its open (substrate-free) form. The final model has been refined at 2.35 Å with a final R of 22.3% (R free ‫ס‬ 23.7%). The structure of the open form is compared to the previously determined structure of the transition state analog complex in the closed form. Classically, the protein would be considered two domain, but dynamic domain (DynDom) analysis shows that most of the differences between the two structures can be considered as the motion between four rigid groups of nonsequential residues. ATP binds near a cluster of positively charged residues of a fixed dynamic domain. The other three dynamic domains close the active site with separate hinge rotations relative to the fixed domain. Several residues of key importance for the induced motion are conserved within the phosphagen kinase family, including creatine kinase. Substantial conformational changes are induced in different parts of the enzyme as intimate interactions are formed with both substrates. Thus, although induced fit occurs in a number of phosphoryl transfer enzymes, the conformational changes in phosphagen kinases appear to be more complicated than in prior examples.

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