Kinetic evidence for a substrate-induced fit in phosphonoacetaldehyde hydrolase catalysis.

Phosphonoacetaldehyde hydrolase (phosphonatase) from Bacillus cereus catalyzes hydrolytic P-C bond cleavage of phosphonoacetaldehyde (Pald) via a Schiff base intermediate formed with Lys53. A single turnover requires binding of Pald to the active site of the core domain, closure of the cap domain containing the Lys53 over the core domain, and dissociation of the products following catalysis. The ligand binding and dissociation steps occur from the "open conformer" (domains are separated and the active site is solvent-exposed), while catalysis occurs from the "closed conformer" (domains are bound together and the active site is sequestered from solvent). To test the hypothesis that bound substrate stabilizes the closed conformer, thus facilitating catalysis, the rates of chemical modification of Lys53 in the presence and absence of inert substrate and/or product analogues were compared. Acetylation of Lys53 with 2,4-dinitrophenylacetate (DNPA) resulted in the loss of enzyme activity. The pseudo-first-order rate constant for inactivation varied with pH. The pH profile of inactivation is consistent with a pK(a) of 9.3 for Lys53. The inhibitors tungstate and vinyl sulfonate, which are known to bind to active site residues comprising the core domain, protected Lys53 from acetylation. These results are consistent with a dynamic equilibrium between the open and closed conformations of phosphonatase and the hypothesis that ligand binding stabilizes the closed conformation required for catalytic turnover.