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 alpha and beta sites to be 29 and 344 microM, respectively, in the absence of a substrate analogue. In its presence, the affinity of the beta site increases substantially (K(d) = 56 microM), while that of the alpha site is not greatly affected (K(d) = 17 microM). 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 (approximately 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.
Hadler
Kieran S
KS
School of Chemistry and Molecular Biosciences, The University of Queensland, St. Lucia, Queensland, 4072, Australia.
Mitić
Natasa
N
Ely
Fernanda
F
Hanson
Graeme R
GR
Gahan
Lawrence R
LR
Larrabee
James A
JA
Ollis
David L
DL
Schenk
Gerhard
G
D055162:Biocatalysis; D001673:Biodegradation, Environmental; D020134:Catalytic Domain; D021902:Enterobacter aerogenes / Q000201:enzymology*; D006860:Hydrogen Bonding; D006868:Hydrolysis; D008280:Magnetics; D008345:Manganese / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010710:Phosphates / Q000378:metabolism; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D010710:Phosphates; D008345:Manganese; D010727:Phosphoric Diester Hydrolases; C038133:glycerophosphodiester phosphodiesterase