Probing the mechanism of enzymatic phosphoryl transfer with a chemical trick.

N substitutions at phosphorus comprise one of the most important classes of reactions in biology. Phosphate diester substitution reactions are catalyzed by nucleases and polymerases and are critical in DNA replication and transcription. Phosphate monoester (phosphoryl transfer) reactions are catalyzed by GTPases, ATPases, protein, and small molecule kinases, protein, and small molecule phosphatases. These enzymes play diverse roles in energy regulation, cell signaling, ion and small molecule transport, and nucleotide synthesis. There have been intensive efforts to try to understand the details of phosphoryl transfer reactions extending from nonenzymatic (or enzyme model) systems to the mechanisms of the enzymatic reactions, as exemplified by the study by Cho et al. in the current issue of PNAS (1). From the many decades of work on small molecules, consensus for a dissociative transition state, akin to an SN1 reaction in organic chemistry, has been reached by most investigators (2, 3). In such a transition state, the bond between the phosphorus and leaving group has largely broken before the formation of a bond between the incoming nucleophile and phosphorus. The role of the nucleophile is diminished, and the formation of a highly reactive metaphosphate-like species is central in a dissociative transition state. This contrasts with the transition state of an associative mechanism, which occurs in phosphate triester substitution reactions, in which a pentavalentlike species is generated. In associative transition states, there is a significant degree of bond formation between the incoming nucleophile and the attacked phosphorus before leaving group departure. Although a full and convincing explanation at the quantum mechanical level has not yet been made as to why dissociative transition states should be preferred for nonenzymatic phosphoryl transfer reactions, the simplest explanation is based on the concept that the negative charges on two of the phosphate oxygens repel incoming nucleophiles and stabilize a metaphosphosphate-like species. More difficult to address is how enzymes catalyze phosphoryl transfer reactions. Enzymatic phosphoryl transfer reactions can occur either by direct transfer of the phosphoryl group to an incoming nucleophile or through the formation of a covalent intermediate (which does not rule out a dissociative transition state). Examples of the latter group of phosphotransferases include alkaline phosphatase, protein tyrosine phosphatases, nucleotide diphosphate kinase, and P-ATPases. In these two-stage reactions, an enzyme nucleophile forms a stable intermediate between the attacked phosphorus while the departing leaving group is expelled. This intermediate is then attacked by an incoming nucleophile, in the case of alkaline phosphatase a water molecule, which regenerates the free enzyme and final product. Phosphotransferases use a variety of enzyme nucleophiles, including: the carboxy group of aspartate (P-ATPases), the thiol of cysteine (protein tyrosine phosphatases), the hydroxy group of serine (alkaline phosphatase), and the imidizaole of histidine (nucleoside diphosphate kinase) (4–7). The linkages involved may or may not be stable in smallmolecule systems, but in the context of the enzymes, they can be quite reactive to facilitate turnover. Consequently, studying their behavior in the structural context of the folded protein may be extremely difficult. Phosphoaspartyl presents a prime example of the problem. The free energy for hydrolysis of phosphoaspartyl hydrolysis is estimated to be several kcal (1 kcal 5 4.18 kJ)ymol more negative than hydrolytic cleavage of the g phosphate of ATP, a very favorable process thermodynamically (8). As a consequence, suitable smallmolecule analogs that mimic the properties of the phosphoaspartyl residues but have greater stability have been sought. BeF3 2 appears to be one such example (9, 10). Cho et al. (1) have shown the general utility of this molecular probe by determining the crystal structure of phosphoserine phosphatase (PSP) bound to BeF3 2