Contribution of the active-site metal cation to the catalytic activity and to the conformational stability of phosphotriesterase: temperature- and pH-dependence.

Phosphotriesterase (PTE) detoxifies nerve agents and organophosphate pesticides. The two zinc cations of the PTE active centre can be substituted by other transition metal cations without loss of activity. Furthermore, metal-substituted PTEs display differences in catalytic properties. A prerequisite for engineering highly efficient mutants of PTE is to improve their thermostability. Isoelectric focusing, capillary electrophoresis and steady-state kinetics analysis were used to determine the contribution of the active-site cations Zn2+, Co2+ or Cd2+ to both the catalytic activity and the conformational stability of the corresponding PTE isoforms. The three isoforms have different pI values (7.2, 7.5 and 7.1) and showed non-superimposable electrophoretic titration curves. The overall structural alterations, causing changes in functional properties, were found to be related to the nature of the bound cation: ionic radius and ion electronegativity correlate with Km and kcat respectively. In addition, the pH-dependent activity profiles of isoforms were different. The temperature-dependent profiles of activity showed maximum activity at T < or =35 degrees C, followed by an activation phase near 45-48 degrees C and then inactivation which was completed at 60 degrees C. Analysis of thermal denaturation of the PTEs provided evidence that the activation phase resulted from a transient intermediate. Finally, at the optimum activity between pH 8 and 9.4, the thermostability of the different PTEs increased as the pH decreased, and the metal cation modulated stability (Zn2+-, Co2+- and Cd2+-PTE showed different T (m) values of 60.5-67 degrees C, 58-64 degrees C and 53-64 degrees C respectively). Requirements for optimum activity of PTE (displayed by Co2+-PTE) and maximum stability (displayed by Zn2+-PTE) were demonstrated.

[1]  Henry Eyring,et al.  Conformation Changes of Proteins , 1954 .

[2]  M. Iwatsubo,et al.  Biosynthetic incorporation of cobalt into yeast alcohol dehydrogenase , 1968, FEBS letters.

[3]  C. Wu,et al.  Role of the intrinsic metal in RNA polymerase from Escherichia coli. In vivo substitution of tightly bound zinc with cobalt. , 1977, Biochemistry.

[4]  F. Raushel,et al.  Purification and properties of the phosphotriesterase from Pseudomonas diminuta. , 1989, The Journal of biological chemistry.

[5]  C. Serdar,et al.  Parathion Hydrolase Gene from Pseudomonas diminuta MG: Subcloning, Complete Nucleotide Sequence, and Expression of the Mature Portion of the Enzyme in Escherichia coli , 1989, Nature Biotechnology.

[6]  V. E. Lewis,et al.  Structure-activity relationships in the hydrolysis of substrates by the phosphotriesterase from Pseudomonas diminuta. , 1989, Biochemistry.

[7]  F. Raushel,et al.  Inactivation of organophosphorus nerve agents by the phosphotriesterase from Pseudomonas diminuta. , 1990, Archives of biochemistry and biophysics.

[8]  Unusual (zig-zag) temperature dependence of the rate constant for irreversible thermoinactivation of hydrophilized enzymes , 1990 .

[9]  F. Raushel,et al.  Limits of diffusion in the hydrolysis of substrates by the phosphotriesterase from Pseudomonas diminuta. , 1991, Biochemistry.

[10]  F. Raushel,et al.  Detoxification of organophosphate pesticides using an immobilized phosphotriesterase from Pseudomonas diminuta , 1991, Biotechnology and bioengineering.

[11]  F. Raushel,et al.  Characterization of the zinc binding site of bacterial phosphotriesterase. , 1992, The Journal of biological chemistry.

[12]  J. M. Sanchez-Ruiz,et al.  Theoretical analysis of Lumry-Eyring models in differential scanning calorimetry. , 1992, Biophysical journal.

[13]  Structural characterization of the divalent cation sites of bacterial phosphotriesterase by 113-Cd NMR spectroscopy , 1993 .

[14]  F. Raushel,et al.  Structural characterization of the divalent cation sites of bacterial phosphotriesterase by 113Cd NMR spectroscopy. , 1993, Biochemistry.

[15]  K. Lai,et al.  Bimetallic binding motifs in organophosphorus hydrolase are important for catalysis and structural organization. , 1994, The Journal of biological chemistry.

[16]  F. Raushel,et al.  Identification of the histidine ligands to the binuclear metal center of phosphotriesterase by site-directed mutagenesis. , 1994, Biochemistry.

[17]  F. Raushel,et al.  Three-dimensional structure of phosphotriesterase: an enzyme capable of detoxifying organophosphate nerve agents. , 1994, Biochemistry.

[18]  F. Raushel,et al.  Metal-substrate interactions facilitate the catalytic activity of the bacterial phosphotriesterase. , 1996, Biochemistry.

[19]  C. Pace,et al.  Organophosphorus hydrolase is a remarkably stable enzyme that unfolds through a homodimeric intermediate. , 1997, Biochemistry.

[20]  W. T. Beaudry,et al.  Enzymatic Hydrolysis of the Chemical Warfare Agent VX and its Neurotoxic Analogues by Organophosphorus Hydrolase , 1997 .

[21]  F. Raushel,et al.  Augmented Hydrolysis of Diisopropyl Fluorophosphate in Engineered Mutants of Phosphotriesterase* , 1997, The Journal of Biological Chemistry.

[22]  J. Cladera,et al.  Effect of nucleotides on the thermal stability and on the deuteration kinetics of the thermophilic F0F1 ATP synthase. , 1997, European journal of biochemistry.

[23]  F. Raushel,et al.  Hydrolysis of Phosphodiesters through Transformation of the Bacterial Phosphotriesterase* , 1998, The Journal of Biological Chemistry.

[24]  F. Raushel,et al.  Stereochemical constraints on the substrate specificity of phosphotriesterase. , 1999, Biochemistry.

[25]  D. Rochu,et al.  Capillary zone electrophoresis with optimized temperature control for studying thermal denaturation of proteins at various pH , 1999, Electrophoresis.

[26]  J. L. Way,et al.  Antagonism of paraoxon intoxication by recombinant phosphotriesterase encapsulated within sterically stabilized liposomes. , 1999, Toxicology and applied pharmacology.

[27]  K. Lai,et al.  Modification of near active site residues in organophosphorus hydrolase reduces metal stoichiometry and alters substrate specificity. , 1999, Biochemistry.

[28]  F. Raushel,et al.  Self-assembly of the binuclear metal center of phosphotriesterase. , 2000, Biochemistry.

[29]  F. Raushel,et al.  Phosphotriesterase: an enzyme in search of its natural substrate. , 2000, Advances in enzymology and related areas of molecular biology.

[30]  F. Raushel,et al.  Structural determinants of the substrate and stereochemical specificity of phosphotriesterase. , 2001, Biochemistry.

[31]  F. Raushel,et al.  High resolution X-ray structures of different metal-substituted forms of phosphotriesterase from Pseudomonas diminuta. , 2001, Biochemistry.

[32]  D. Rochu,et al.  Thermal stability of acetylcholinesterase from Bungarus fasciatus venom as investigated by capillary electrophoresis. , 2001, Biochimica et biophysica acta.

[33]  F. Raushel,et al.  Enhancement, relaxation, and reversal of the stereoselectivity for phosphotriesterase by rational evolution of active site residues. , 2001, Biochemistry.

[34]  Pressure- and heat-induced inactivation of butyrylcholinesterase: evidence for multiple intermediates and the remnant inactivation process. , 2001, The Biochemical journal.

[35]  Wilfred Chen,et al.  Bacterial Cell Surface Display of Organophosphorus Hydrolase for Selective Screening of Improved Hydrolysis of Organophosphate Nerve Agents , 2002, Applied and Environmental Microbiology.

[36]  F. Raushel.  Bacterial detoxification of organophosphate nerve agents. , 2002, Current opinion in microbiology.

[37]  D. Rochu,et al.  The wild type bacterial Co(2+)/Co(2+)-phosphotriesterase shows a middle-range thermostability. , 2002, Biochimica et biophysica acta.

[38]  F. Raushel,et al.  Enhanced degradation of chemical warfare agents through molecular engineering of the phosphotriesterase active site. , 2003, Journal of the American Chemical Society.