Structural flexibility enhances the reactivity of the bioremediator glycerophosphodiesterase by fine-tuning its mechanism of hydrolysis

The glycerophosphodiesterase from Enterobacter aerogenes (GpdQ) belongs to the family of binuclear metallohydrolases and has attracted recent attention due to its potential in bioremediation. Formation of a catalytically competent binuclear center is promoted by the substrate (Hadler et al. J. Am. Chem. Soc. 2008, 130, 14129). Using the paramagnetic properties of Mn(II), we estimated the K_d values for the metal ions in the α and β sites to be 29 and 344 μM, respectively, in the absence of a substrate analogue. In its presence, the affinity of the β site increases substantially (K_d ) 56 μM), while that of the R site is not greatly affected (K_d ) 17 μM). Stopped-flow fluorescence measurements identified three distinct phases in the catalytic turnover, associated with the initial binding of substrate to the active site (k_obs1), the assembly of a catalytically active binuclear center (k_obs2), and subsequent slower structural rearrangements to optimize catalysis (k_obs3). These three phases depend on the concentration of substrate ([S]), with k_obs1 and k _obs2 reaching maximum values at high [S] (354 and 38 s_-1, respectively), whereas k_obs3 is reduced as [S] is increased. The k_cat for the hydrolysis of the substrate bis(para-nitrophenyl) phosphate (∼1 s_-1) gradually increases from the moment of initiating the reaction, reaching a maximum when the structural change associated with k_obs3 is complete. This structural change is mediated via an extensive hydrogen-bond network that connects the coordination sphere with the substrate binding pocket, as demonstrated by mutation of two residues in this network (His81 and His217). The identities of both the substrate and the metal ion also affect interactions within this H-bond network, thus leading to some mechanistic variations. Overall, the mechanism employed by GpdQ is a paradigm of a substrate- and metal-ion-induced fit to optimize catalysis.