Cadmium(II) complexes of the glycerophosphodiester-degrading enzyme GpdQ and a biomimetic N,O ligand

Abstract

The glycerophosphodiester-degrading enzyme GpdQ from Enterobacter aerogenes is a promising bioremediator owing to its ability to degrade some organophosphate pesticides and diester products originating from the hydrolysis of nerve agents such as VX. Here, the cadmium derivative of GpdQ was prepared by reconstituting the apoenzyme. Catalytic measurements with (Cd(2+))(2)-GpdQ and the phosphodiester substrate bis(4-nitrophenyl)phosphate yield k(cat) = 15 s(-1). The pK(a) of 9.4, determined from the pH dependence of the catalytic activity, implicates a hydroxide ligand as the catalytic nucleophile. Also prepared was the cadmium-containing biomimetic [Cd(2)((HP)(2)B)(OAc)(2)(OH(2))](PF(6)) (where (HP)(2)B is [2,6-bis([(2-pyridylmethyl)(2-hydroxyethyl)amino]methyl)-4-methylphenol]), which mimics the asymmetry of the metal ion coordination in the active site of GpdQ. The phosphoesterase-like activity of [Cd(2)((HP)(2)B)(OAc)(2)(OH(2))](PF(6)) was studied using the substrate bis(2,4-dinitrophenyl)phosphate, yielding a kinetically relevant pK(a) of 8.9, with k(cat) = 0.004 s(-1). In summary, the model is both an adequate structural and a reasonable functional mimic of GpdQ.

Spectroscopic and mechanistic studies of dinuclear metallohydrolases and their biomimetic complexes

“…80,138 This effect is analogous to the effect of the parasubstituents seen in the di-Zn(II) complexes. The latter complex with the same donor atom set as the former but a methyl group in para-position of the bridging phenolic oxygen, exhibited a pK a around 9,80 suggesting that the ethyl ester has an effect on the polarization of the nucleophile, shifting the catalytically relevant pK a . Peralta et al reported a linear correlation between the pK a values attributed to deprotonation of the Fe(III)-bound water for complexes with different substituents, methyl, H, Br and NO 2 respectively.…”

“…The in vivo metal ion content in GpdQ has, to date, not been determined, although studies using anomalous scattering experiments suggest Fe(II) bound in the α-site. 75 Catalytic activity can be reconstituted with a variety of divalent metal ions including Co(II), Zn(II), Fe(II), Mn(II) and Cd(II), 75,79,80 and heterodinuclear derivatives of GpdQ have been reported in an attempt to explore the in vivo metal ion content. 75,81 As Co(II) serves as an excellent spectroscopic probe, the Co(II)Co(II) derivative is the most well studied metal derivative.…”

“…In the same work, studies of Cd 2 -GpdQ and the phosphodiester substrate bis-(4-nitrophenyl)phosphate (BPNPP) resulted in a k cat of 15 s −1 and a catalytically relevant pK a of 9.4. 80 For both the biomimetic and the enzyme, a terminal hydroxide is implicated as the catalytic nucleophile. Another mechanistic study of a Cd(II)-substituted phosphoesterase has been reported by Ely et al 71 The kinetically relevant pK a (∼4-5) for Cd 2 -OpdA (organophosphate-degrading agent) in the hydrolysis of ethyl paraoxon suggested a bridging hydroxide as the nucleophile.…”

Structural Flexibility Enhances the Reactivity of the Bioremediator Glycerophosphodiesterase by Fine-Tuning Its Mechanism of Hydrolysis

“…The Co(II)-and Cd(II)-derivatives of GpdQ were prepared as previously described. 10, 25 The Mn(II) form was prepared by adding a 99.9% pure metal chloride solution to the apoenzyme (prepared as described elsewhere 10 ).…”

Immobilization of the enzyme GpdQ on magnetite nanoparticles for organophosphate pesticide bioremediation

“…Although the metal ion content of GpdQ in vivo is currently unknown, it was initially proposed, on the basis of spectroscopic and crystallographic results, that the native metal ion composition was Fe(II)Zn(II). The reactivity of apo-GpdQ can, however, be readily reconstituted with Co(II), Mn(II), Fe(II), Zn(II) and even Cd(II) [15,16]. As Co(II) serves as an excellent spectroscopic probe, the Co(II)Co(II) derivative is the best studied metal derivative.…”