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Crystal structures of a purple acid phosphatase, representing different steps of this enzyme's catalytic cycle.

Schenk G, Elliott TW, Leung E, Carrington LE, Mitić N, Gahan LR, Guddat LW - BMC Struct. Biol. (2008)

Bottom Line: Importantly, both metal ions are five-coordinate in this structure, with only one nucleophilic mu-hydroxide present in the metal-bridging position.The fluoride-bound structure provides visual support for an activation mechanism for this mu-hydroxide whereby substrate binding induces a shift of this bridging ligand towards the divalent metal ion, thus increasing its nucleophilicity.In combination with kinetic, crystallographic and spectroscopic data these structures of red kidney bean purple acid phosphatase facilitate the proposal of a comprehensive eight-step model for the catalytic mechanism of purple acid phosphatases in general.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Molecular and Microbial Sciences, The University of Queensland, St, Lucia, QLD 4072, Australia. schenk@uq.edu.au

ABSTRACT

Background: Purple acid phosphatases belong to the family of binuclear metallohydrolases and are involved in a multitude of biological functions, ranging from bacterial killing and bone metabolism in animals to phosphate uptake in plants. Due to its role in bone resorption purple acid phosphatase has evolved into a promising target for the development of anti-osteoporotic chemotherapeutics. The design of specific and potent inhibitors for this enzyme is aided by detailed knowledge of its reaction mechanism. However, despite considerable effort in the last 10 years various aspects of the basic molecular mechanism of action are still not fully understood.

Results: Red kidney bean purple acid phosphatase is a heterovalent enzyme with an Fe(III)Zn(II) center in the active site. Two new structures with bound sulfate (2.4 A) and fluoride (2.2 A) provide insight into the pre-catalytic phase of its reaction cycle and phosphorolysis. The sulfate-bound structure illustrates the significance of an extensive hydrogen bonding network in the second coordination sphere in initial substrate binding and orientation prior to hydrolysis. Importantly, both metal ions are five-coordinate in this structure, with only one nucleophilic mu-hydroxide present in the metal-bridging position. The fluoride-bound structure provides visual support for an activation mechanism for this mu-hydroxide whereby substrate binding induces a shift of this bridging ligand towards the divalent metal ion, thus increasing its nucleophilicity.

Conclusion: In combination with kinetic, crystallographic and spectroscopic data these structures of red kidney bean purple acid phosphatase facilitate the proposal of a comprehensive eight-step model for the catalytic mechanism of purple acid phosphatases in general.

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Stereodiagram of the active site of the rkbPAP-fluoride complex. Fo-Fc electron density for the bridge is overlayed. The fluoride replaces the hydroxide in the bridging position. General legend: Fe(III) is in tan, Zn(II) in grey, sodium in purple, fluoride in cyan, carbon in green, oxygen in red, nitrogen in blue and sulphur in orange. Hydrogen bonds and other contacts are shown as dashed lines.
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Figure 4: Stereodiagram of the active site of the rkbPAP-fluoride complex. Fo-Fc electron density for the bridge is overlayed. The fluoride replaces the hydroxide in the bridging position. General legend: Fe(III) is in tan, Zn(II) in grey, sodium in purple, fluoride in cyan, carbon in green, oxygen in red, nitrogen in blue and sulphur in orange. Hydrogen bonds and other contacts are shown as dashed lines.

Mentions: rkbPAP incubated with fluoride crystallized in a different space group and with different unit cell parameters compared to the sulfate complex (Table 2). Two dimers in the asymmetric unit are observed yielding four copies of the active site. In these structures the metal ions are on average 3.5 Å apart, a distance significantly larger than that observed in any of the other rkbPAP structures [40,41]. A small sphere of electron density is observed between the two metal ions (Figure 4), in a position similar, but not identical, to that of the μ-hydroxo in the rkbPAP-sulfate complex. The center of the bridging electron density in the rkbPAP-F structure is 2.5 Å and 2.1 Å from Fe(III) and Zn(II), respectively (Table 1). In contrast, the bridging electron density in rkbPAP-SO4 is, respectively, 2.1 Å and 2.3 Å from Fe(III) and Zn(II). Thus, the bridging ligand is shifted 0.2 Å closer to the Zn(II) and 0.4 Å further away from the Fe(III); its distance to the carbonyl oxygen of His323 is 3.6 Å, compared to 3.1 Å observed in the rkbPAP-SO4 structure (Figure 2). These differences indicate that fluoride rather than μ-hydroxide acts as the bridging ligand, as anticipated from kinetic and spectroscopic data collected for PAPs from pig, human and red kidney bean [15,49,50], and other binuclear metallohydrolases [51]. Additional electron densities in the second coordination sphere, close to the fluoride, are tentatively assigned to a sodium ion and a sulfate ion (see Methods section). The sodium ion may offset the charge of the metal bridging fluoride group, and possibly prevents cations (i.e. sulfate, acetate) from binding to the second coordination sphere.


Crystal structures of a purple acid phosphatase, representing different steps of this enzyme's catalytic cycle.

Schenk G, Elliott TW, Leung E, Carrington LE, Mitić N, Gahan LR, Guddat LW - BMC Struct. Biol. (2008)

Stereodiagram of the active site of the rkbPAP-fluoride complex. Fo-Fc electron density for the bridge is overlayed. The fluoride replaces the hydroxide in the bridging position. General legend: Fe(III) is in tan, Zn(II) in grey, sodium in purple, fluoride in cyan, carbon in green, oxygen in red, nitrogen in blue and sulphur in orange. Hydrogen bonds and other contacts are shown as dashed lines.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2267794&req=5

Figure 4: Stereodiagram of the active site of the rkbPAP-fluoride complex. Fo-Fc electron density for the bridge is overlayed. The fluoride replaces the hydroxide in the bridging position. General legend: Fe(III) is in tan, Zn(II) in grey, sodium in purple, fluoride in cyan, carbon in green, oxygen in red, nitrogen in blue and sulphur in orange. Hydrogen bonds and other contacts are shown as dashed lines.
Mentions: rkbPAP incubated with fluoride crystallized in a different space group and with different unit cell parameters compared to the sulfate complex (Table 2). Two dimers in the asymmetric unit are observed yielding four copies of the active site. In these structures the metal ions are on average 3.5 Å apart, a distance significantly larger than that observed in any of the other rkbPAP structures [40,41]. A small sphere of electron density is observed between the two metal ions (Figure 4), in a position similar, but not identical, to that of the μ-hydroxo in the rkbPAP-sulfate complex. The center of the bridging electron density in the rkbPAP-F structure is 2.5 Å and 2.1 Å from Fe(III) and Zn(II), respectively (Table 1). In contrast, the bridging electron density in rkbPAP-SO4 is, respectively, 2.1 Å and 2.3 Å from Fe(III) and Zn(II). Thus, the bridging ligand is shifted 0.2 Å closer to the Zn(II) and 0.4 Å further away from the Fe(III); its distance to the carbonyl oxygen of His323 is 3.6 Å, compared to 3.1 Å observed in the rkbPAP-SO4 structure (Figure 2). These differences indicate that fluoride rather than μ-hydroxide acts as the bridging ligand, as anticipated from kinetic and spectroscopic data collected for PAPs from pig, human and red kidney bean [15,49,50], and other binuclear metallohydrolases [51]. Additional electron densities in the second coordination sphere, close to the fluoride, are tentatively assigned to a sodium ion and a sulfate ion (see Methods section). The sodium ion may offset the charge of the metal bridging fluoride group, and possibly prevents cations (i.e. sulfate, acetate) from binding to the second coordination sphere.

Bottom Line: Importantly, both metal ions are five-coordinate in this structure, with only one nucleophilic mu-hydroxide present in the metal-bridging position.The fluoride-bound structure provides visual support for an activation mechanism for this mu-hydroxide whereby substrate binding induces a shift of this bridging ligand towards the divalent metal ion, thus increasing its nucleophilicity.In combination with kinetic, crystallographic and spectroscopic data these structures of red kidney bean purple acid phosphatase facilitate the proposal of a comprehensive eight-step model for the catalytic mechanism of purple acid phosphatases in general.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Molecular and Microbial Sciences, The University of Queensland, St, Lucia, QLD 4072, Australia. schenk@uq.edu.au

ABSTRACT

Background: Purple acid phosphatases belong to the family of binuclear metallohydrolases and are involved in a multitude of biological functions, ranging from bacterial killing and bone metabolism in animals to phosphate uptake in plants. Due to its role in bone resorption purple acid phosphatase has evolved into a promising target for the development of anti-osteoporotic chemotherapeutics. The design of specific and potent inhibitors for this enzyme is aided by detailed knowledge of its reaction mechanism. However, despite considerable effort in the last 10 years various aspects of the basic molecular mechanism of action are still not fully understood.

Results: Red kidney bean purple acid phosphatase is a heterovalent enzyme with an Fe(III)Zn(II) center in the active site. Two new structures with bound sulfate (2.4 A) and fluoride (2.2 A) provide insight into the pre-catalytic phase of its reaction cycle and phosphorolysis. The sulfate-bound structure illustrates the significance of an extensive hydrogen bonding network in the second coordination sphere in initial substrate binding and orientation prior to hydrolysis. Importantly, both metal ions are five-coordinate in this structure, with only one nucleophilic mu-hydroxide present in the metal-bridging position. The fluoride-bound structure provides visual support for an activation mechanism for this mu-hydroxide whereby substrate binding induces a shift of this bridging ligand towards the divalent metal ion, thus increasing its nucleophilicity.

Conclusion: In combination with kinetic, crystallographic and spectroscopic data these structures of red kidney bean purple acid phosphatase facilitate the proposal of a comprehensive eight-step model for the catalytic mechanism of purple acid phosphatases in general.

Show MeSH