Magnesium in the active site of Escherichia coli alkaline phosphatase is important for both structural stabilization and catalysis

Janeway, Claude M.; Xu, Xu; Murphy, Jennifer E.; Chaidaroglou, Antigoni; Kantrowitz, Evan R.

doi:10.1021/bi00057a026

Cited by 85 publications

(88 citation statements)

References 43 publications

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“…A dual role for metal ions in both structural stabilization and catalysis has also been observed in other proteins (38 -41). For instance, the presence of magnesium in the active site of the Escherichia coli alkaline phosphatase has been shown to be important for both catalytic activity and stability of the enzyme (38).…”

Section: Discussionmentioning

confidence: 99%

Effect of Metal Ion Binding on the Structural Stability of the Hepatitis C Virus RNA Polymerase

Benzaghou

Bougie

Bisaillon

2004

Journal of Biological Chemistry

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The RNA polymerase activity of the hepatitis C virus, a major human pathogen, has previously been shown to be supported by metal ions. In the present study, we report a systematic analysis of the effect of metal ion binding on the structural stability of the hepatitis C virus RNA polymerase. Chemical and thermal denaturation assays revealed that the stability of the protein is increased significantly in the presence of metal ions. Structural analyses clearly established that metal ion binding increases hydrophobic exposure on the RNA polymerase surface. Furthermore, our denaturation studies, coupled with polymerization assays, demonstrate that the active site region of the polymerase is more sensitive to chemical denaturant than other structural scaffolds. We also report the first detailed study of the thermodynamic parameters involved in the interaction between the hepatitis C virus RNA polymerase and metal ions. Finally, a mutational analysis was also performed to investigate the importance of Asp 220 , Asp 318 , and Asp 319 for metal ion binding. This mutational study underscores a strict requirement for each of the residues for metal binding, indicating that the active center of the HCV RNA polymerase is intolerant to virtually any perturbations of the metal coordination sphere, thereby highlighting the critical role of the enzymebound metal ions. Overall, our results indicate that metal ions play a dual modulatory role in the RNA polymerase reaction by promoting both a favorable geometry of the active site for catalysis and by increasing the structural stability of the enzyme.Recent estimates indicate that more than 170 million people worldwide are infected with the hepatitis C virus (HCV) 1 (1). It is estimated that about 80% of patients with acute HCV infection will progress to chronic hepatitis. Of these, 20% will develop cirrhosis, and 1-5% will develop hepatocellular carcinoma (2-5). There is thus an urgent need for the development of antiviral drugs aimed at inhibiting this pathogen.The HCV nonstructural 5B protein (NS5B) has been shown to be an RNA-dependent RNA polymerase (6 -11). The protein contains characteristic motifs, such as the GDD motif, shared by RNA-dependent RNA polymerases, and is believed to be responsible for the genome replication of HCV (12). The NS5B protein has been studied extensively during the past few years because it is one of the major targets for the development of antiviral drugs (7,(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25). The enzyme can utilize a wide range of RNA molecules as template, although it appears to prefer certain homopolyribonucleotides (26). By itself, NS5B appears to lack specificity for HCV RNA and displays activity on heterologous nonviral RNA (3). This lack of specificity for HCV RNA supports the notion that additional viral or cellular factors are required for specific recognition of the viral replication signal. The HCV RNA polymerase activity has been shown to be supported by both magnesium and manganese ions (7, 13, 21-25). However, the re...

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Section: Discussionmentioning

confidence: 99%

Effect of Metal Ion Binding on the Structural Stability of the Hepatitis C Virus RNA Polymerase

Benzaghou

Bougie

Bisaillon

2004

Journal of Biological Chemistry

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“…Studies of E. coli AP concerning the secondary ligands of Mg(II) (D153 and K328 [mature E. coli AP sequence numbering]) have led to the conclusion that replacement of these residues with histidine results in the transformation of an Mg(II)-binding site into a Zn(II)-binding site. The affinity between Mg(II) and its binding site became lower for the (D153H, K328H) mutant AP, and optimal activity was recovered by the addition of Mg(II) (20,24,33,34). The residues D153 and K328 in E. coli AP correspond to the residues H108 and H228 in P. abyssi AP.…”

Section: Discussionmentioning

confidence: 99%

Characterization of a Highly Thermostable Alkaline Phosphatase from the EuryarchaeonPyrococcus abyssi

Zappa

Rolland

Flament

et al. 2001

Appl Environ Microbiol

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This work reports the first isolation and characterization of an alkaline phosphatase (AP) from a hyperthermophilic archaeon. An AP gene from Pyrococcus abyssi, a euryarchaeon isolated from a deep-sea hydrothermal vent, was cloned and the enzyme expressed in Escherichia coli. Analysis of the sequence showed conservation of the active site and structural elements of the E. coli AP. The recombinant AP was purified and characterized. Monomeric and homodimeric active forms were detected, with a monomer molecular mass of 54 kDa. Apparent optimum pH and temperature were estimated at 11.0 and 70°C, respectively. Thus far, P. abyssi AP has been demonstrated to be the most thermostable AP, with half-lives at 100 and 105°C of 18 and 5 h, respectively. Enzyme activity was found to be dependent on divalent cations: metal ion chelators inhibited activity, whereas the addition of exogenous Mg(II), Zn(II), and Co(II) increased activity. The enzyme was inhibited by inorganic phosphate, but not by molybdate and vanadate. Strong inhibitory effects were observed in the presence of thiolreducing agents, although cysteine residues of the P. abyssi AP were not found to be incorporated within intraor interchain disulfide bonds. In addition, P. abyssi AP was demonstrated to dephosphorylate linear DNA fragments with dephosphorylation efficiencies of 93.8 and 84.1% with regard to cohesive and blunt ends, respectively.

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“…For example, replacement of Asp-153 by His resulted in an enzyme with an altered pH profile and enhanced activity in the presence of excess Mg2+ (Janeway et al, 1993). The X-ray structure of this enzyme, without excess Mg2+, indicated that the enzyme exists with Zn2+ in the Mg site .…”

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confidence: 99%

Escherichia coli alkaline phosphatase: X‐ray structural studies of a mutant enzyme (His‐412 → Asn) at one of the catalytically important zinc binding sites

Tibbitts

Kantrowitz

1995

Protein Science

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The X-ray structure of a mutant version of Escherichia coli alkaline phosphatase (H412N) in which His-412 was replaced by Asn has been determined at both low (-Zn) and high (+Zn) concentrations of zinc. In the wild-type structure, His-412 is a direct ligand to one of the two catalytically critical zinc atoms (Zn,) in the active site. Characterization of the H412N enzyme in solution revealed that the mutant enzyme required high concentrations of zinc for maximal activity and for high substrate and phosphate affinity (Ma L, Kantrowitz ER, 1994, JBiol Chem 269:31614-31619). The H412N enzyme was also inhibited by Tris, in contrast to the wild-type enzyme, which is activated more than twofold by 1 M Tris. To understand these kinetic properties at the molecular level, the structure of the H412N(+Zn) enzyme was refined to an R-factor of 0.174 at 2.2 A resolution, and the structure of the H412N(-Zn) enzyme was refined to an R-factor of 0.166 at a resolution of 2.6 A. Both indicated that the Asn residue substituted for His-412 did not coordinate well to Zn,. In the H412N(-Zn) structure, the Zn, site had very low occupancy and the phosphate was shifted by 1.8 A from its position in the wild-type structure. The Mg binding site was also affected by the substitution of Asn for His-412. Both structures of the H412N enzyme also revealed a surface-accessible cavity near the Zn, site that may serve as a binding site for Tris. The binding of Tris at the Zn, site may block Zn2+ binding at this site, which would account for the reduced affinity of the H412N enzyme for substrate as well as its reduced catalytic efficiency in the presence of Tris. Thus, the replacement of His-412 with Asn affects all three metal binding sites, suggesting that the relative positions of the metals bound at the active site of E. coli alkaline phosphatase are strongly linked and critical for efficient catalysis.Keywords: metal binding site; metalloenzymes; protein structure-function; site-specific mutagenesis; X-ray diffraction Alkaline phosphatase (EC 3.1.3.1) is a nonspecific phosphomonoesterase found in most organisms (Coleman, 1992) that catalyzes the hydrolysis reaction via a phosphoseryl intermediate (Schwartz & Lipmann, 1961;Engstrom, 1962) Abbreviations: H412N, the mutant alkaline phosphatase that has His-412 replaced by Asn; H412N(-Zn), the structure of the H412N enzyme determined with crystals that did not have added zinc in the stabilization buffer; H412N(+Zn), the structure of the H412N enzyme determined with crystals soaked in stabilization buffer containing 10 mM zinc chloride; Pi, inorganic phosphate; RMSD, RMS displacement; Zn,, Znz, and Mg sites correspond to the M1, M2, and M3 sites as identified in the X-ray structure of the wild-type enzyme (Sowadski et al., 1983); Zn, ,,the Zn2+ ion bound in the M1 site of alkaline phosphatase; Znz, the Zn2+ ion bound in the M2 site of alkaline phosphatase; Mg, the Mg2+ ion bound in the M3 site of alkaline phosphatase.

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Magnesium in the active site of Escherichia coli alkaline phosphatase is important for both structural stabilization and catalysis

Cited by 85 publications

References 43 publications

Effect of Metal Ion Binding on the Structural Stability of the Hepatitis C Virus RNA Polymerase

Effect of Metal Ion Binding on the Structural Stability of the Hepatitis C Virus RNA Polymerase

Characterization of a Highly Thermostable Alkaline Phosphatase from the EuryarchaeonPyrococcus abyssi

Escherichia coli alkaline phosphatase: X‐ray structural studies of a mutant enzyme (His‐412 → Asn) at one of the catalytically important zinc binding sites

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