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Identification of a Histidine Metal Ligand in the argE-Encoded N-Acetyl-L-Ornithine Deacetylase from Escherichia coli.

McGregor WC, Gillner DM, Swierczek SI, Liu D, Holz RC - Springerplus (2013)

Bottom Line: The H355A, H355K, H80A, and H80K mutant enzymes of the argE-encoded N-acetyl-L-ornithine deacetylase (ArgE) from Escherichia coli were prepared, however, only the H355A enzyme was found to be soluble.Kinetic analysis of the Co(II)-loaded H355A exhibited activity levels that were 380-fold less than Co(II)-loaded WT ArgE.A three-dimensional homology model of ArgE was generated using the X-ray crystal structure of the dapE-encoded N-succinyl-L,L-diaminopimelic acid desuccinylase (DapE) from Haemophilus influenzae confirming the assignment of H355 as well as H80 as active site ligands.

View Article: PubMed Central - PubMed

Affiliation: The Department of Applied Sciences and Mathematics, College of Technology and Innovation, Arizona State University, Mesa, AZ 85212 USA.

ABSTRACT
The H355A, H355K, H80A, and H80K mutant enzymes of the argE-encoded N-acetyl-L-ornithine deacetylase (ArgE) from Escherichia coli were prepared, however, only the H355A enzyme was found to be soluble. Kinetic analysis of the Co(II)-loaded H355A exhibited activity levels that were 380-fold less than Co(II)-loaded WT ArgE. Electronic absorption spectra of Co(II)-loaded H355A-ArgE indicate that the bound Co(II) ion resides in a distorted, five-coordinate environment and Isothermal Titration Calorimetry (ITC) data for Zn(II) binding to the H355A enzyme provided a dissociation constant (K d) of 39 μM. A three-dimensional homology model of ArgE was generated using the X-ray crystal structure of the dapE-encoded N-succinyl-L,L-diaminopimelic acid desuccinylase (DapE) from Haemophilus influenzae confirming the assignment of H355 as well as H80 as active site ligands.

No MeSH data available.


Related in: MedlinePlus

Plot of Δϵ560vs. the concentration of free Co(II) ions in solution for Co(II) titration into apo-H355A ArgE (50 mM KH2PO4buffer, pH 7.5).
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Fig5: Plot of Δϵ560vs. the concentration of free Co(II) ions in solution for Co(II) titration into apo-H355A ArgE (50 mM KH2PO4buffer, pH 7.5).

Mentions: where p is the number of sites for which interaction with Co(II) is governed by the intrinsic dissociation constant Kd, Cs is the free metal concentration and, r is the binding function calculated by conversion of the fractional saturation (fa) (Winzor and Sawyer 1995). Values for Kd and p were obtained by fitting these data via an iterative process that allowed Kd and p to vary (Figure 5). The best fits obtained for Co(II) binding to the H355A ArgE provided a p value of 0.9 and a Kd value of 1.6 ± 0.3 μM, which is 4-fold larger than that observed for WT ArgE (Kd = 0.4 μM) (McGregor et al. 2005) consistent with a loss of an active site ligand. Since alteration of H355 will greatly perturb and potentially alter the ability of metal binding, this Kd value likely corresponds to a single metal binding event. Moreover, the Kd value obtained for Co(II) binding to H355A is ~4 times stronger than that obtained for Zn(II) binding to WT ArgE. This difference is typically related to Zn(II)’s preference for four or five-coordinate geometries vs. Co(II)’s preference for five or six-coordinate structures (Holz 2002). These data, taken together with the lack of observed activity for H355A ArgE in the presence of Zn(II), suggest that H355 is in fact an active site ligand.Figure 5


Identification of a Histidine Metal Ligand in the argE-Encoded N-Acetyl-L-Ornithine Deacetylase from Escherichia coli.

McGregor WC, Gillner DM, Swierczek SI, Liu D, Holz RC - Springerplus (2013)

Plot of Δϵ560vs. the concentration of free Co(II) ions in solution for Co(II) titration into apo-H355A ArgE (50 mM KH2PO4buffer, pH 7.5).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4320195&req=5

Fig5: Plot of Δϵ560vs. the concentration of free Co(II) ions in solution for Co(II) titration into apo-H355A ArgE (50 mM KH2PO4buffer, pH 7.5).
Mentions: where p is the number of sites for which interaction with Co(II) is governed by the intrinsic dissociation constant Kd, Cs is the free metal concentration and, r is the binding function calculated by conversion of the fractional saturation (fa) (Winzor and Sawyer 1995). Values for Kd and p were obtained by fitting these data via an iterative process that allowed Kd and p to vary (Figure 5). The best fits obtained for Co(II) binding to the H355A ArgE provided a p value of 0.9 and a Kd value of 1.6 ± 0.3 μM, which is 4-fold larger than that observed for WT ArgE (Kd = 0.4 μM) (McGregor et al. 2005) consistent with a loss of an active site ligand. Since alteration of H355 will greatly perturb and potentially alter the ability of metal binding, this Kd value likely corresponds to a single metal binding event. Moreover, the Kd value obtained for Co(II) binding to H355A is ~4 times stronger than that obtained for Zn(II) binding to WT ArgE. This difference is typically related to Zn(II)’s preference for four or five-coordinate geometries vs. Co(II)’s preference for five or six-coordinate structures (Holz 2002). These data, taken together with the lack of observed activity for H355A ArgE in the presence of Zn(II), suggest that H355 is in fact an active site ligand.Figure 5

Bottom Line: The H355A, H355K, H80A, and H80K mutant enzymes of the argE-encoded N-acetyl-L-ornithine deacetylase (ArgE) from Escherichia coli were prepared, however, only the H355A enzyme was found to be soluble.Kinetic analysis of the Co(II)-loaded H355A exhibited activity levels that were 380-fold less than Co(II)-loaded WT ArgE.A three-dimensional homology model of ArgE was generated using the X-ray crystal structure of the dapE-encoded N-succinyl-L,L-diaminopimelic acid desuccinylase (DapE) from Haemophilus influenzae confirming the assignment of H355 as well as H80 as active site ligands.

View Article: PubMed Central - PubMed

Affiliation: The Department of Applied Sciences and Mathematics, College of Technology and Innovation, Arizona State University, Mesa, AZ 85212 USA.

ABSTRACT
The H355A, H355K, H80A, and H80K mutant enzymes of the argE-encoded N-acetyl-L-ornithine deacetylase (ArgE) from Escherichia coli were prepared, however, only the H355A enzyme was found to be soluble. Kinetic analysis of the Co(II)-loaded H355A exhibited activity levels that were 380-fold less than Co(II)-loaded WT ArgE. Electronic absorption spectra of Co(II)-loaded H355A-ArgE indicate that the bound Co(II) ion resides in a distorted, five-coordinate environment and Isothermal Titration Calorimetry (ITC) data for Zn(II) binding to the H355A enzyme provided a dissociation constant (K d) of 39 μM. A three-dimensional homology model of ArgE was generated using the X-ray crystal structure of the dapE-encoded N-succinyl-L,L-diaminopimelic acid desuccinylase (DapE) from Haemophilus influenzae confirming the assignment of H355 as well as H80 as active site ligands.

No MeSH data available.


Related in: MedlinePlus