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PDBsum entry 1alh

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Hydrolase (phosphoric monoester) PDB id
1alh
Jmol
Contents
Protein chains
446 a.a. *
Ligands
PO4 ×2
SO4 ×2
Metals
_ZN ×2
Waters ×888
* Residue conservation analysis

References listed in PDB file
Key reference
Title Kinetics and crystal structure of a mutant escherichia coli alkaline phosphatase (asp-369-->Asn): a mechanism involving one zinc per active site.
Authors T.T.Tibbitts, X.Xu, E.R.Kantrowitz.
Ref. Protein Sci, 1994, 3, 2005-2014. [DOI no: 10.1002/pro.5560031113]
PubMed id 7703848
Note In the PDB file this reference is annotated as "TO BE PUBLISHED". The citation details given above were identified by an automated search of PubMed on title and author names, giving a percentage match of 96%.
Abstract
Using site-directed mutagenesis, an aspartate side chain involved in binding metal ions in the active site of Escherichia coli alkaline phosphatase (Asp-369) was replaced, alternately, by asparagine (D369N) and by alanine (D369A). The purified mutant enzymes showed reduced turnover rates (kcat) and increased Michaelis constants (Km). The kcat for the D369A enzyme was 5,000-fold lower than the value for the wild-type enzyme. The D369N enzyme required Zn2+ in millimolar concentrations to become fully active; even under these conditions the kcat measured for hydrolysis of p-nitrophenol phosphate was 2 orders of magnitude lower than for the wild-type enzyme. Thus the kcat/Km ratios showed that catalysis is 50 times less efficient when the carboxylate side chain of Asp-369 is replaced by the corresponding amide; and activity is reduced to near nonenzymic levels when the carboxylate is replaced by a methyl group. The crystal structure of D369N, solved to 2.5 A resolution with an R-factor of 0.189, showed vacancies at 2 of the 3 metal binding sites. On the basis of the kinetic results and the refined X-ray coordinates, a reaction mechanism is proposed for phosphate ester hydrolysis by the D369N enzyme involving only 1 metal with the possible assistance of a histidine side chain.
Figure 6.
Fig. 6. Stereo pair showing the electron density of the anion binding site ound in D369N alkaline phosphatase. The ligand at this site, which may be ei- phosphate or sulfate, was modeled assulfatedurigthe refinement. Fig- 6,7,and 8 were produced sing the program SETOR (Evans, 1993).
Figure 8.
Fig. 8. pair comparing the posi- tions of side chains, metals, andphos- phate in the active sites of the D369N enzyme (thick lines) and the wild-type enzyme (thin lines). At the I site, zinc is bound close to he same location in both enzymes (crosses), but phosphate (PO,) is more exposed to the surface in the D369N structure. At the M2 site, zinc is bound n the wild-type (cross) but not theutant enzyme. This permits SI02 to move slightly closer to H370 and the asparagineintroduce at position 369 (D369N). The M3 site normally contains Mgz+ (cross) with 3 water molecules (not shown) in the wild-type enzyme; in the mutant enzyme this space is partially filled y 1 water molecule (not shown), the carboxylate side chain of D5 1, and the#-aminogroup of K328. The 2 sets of atomiccordinates were aligned using Quanta to minimize the RMSD of the Cor atomsbefore making this omparison.
The above figures are reprinted from an Open Access publication published by the Protein Society: Protein Sci (1994, 3, 2005-2014) copyright 1994.
Secondary reference #1
Title Reaction mechanism of alkaline phosphatase based on crystal structures. Two-Metal ion catalysis.
Authors E.E.Kim, H.W.Wyckoff.
Ref. J Mol Biol, 1991, 218, 449-464.
PubMed id 2010919
Abstract
PROCHECK
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