4KM4: E. coli alkaline phosphatase mutant S102G/R166S in complex with inorganic phosphate

Enzymes stabilize transition states of reactions while limiting binding to ground states, as is generally required for any catalyst. Alkaline Phosphatase (AP) and other nonspecific phosphatases are some of Nature's most impressive catalysts, achieving preferential transition state over ground state stabilization of more than 10(2)(2)-fold while utilizing interactions with only the five atoms attached to the transferred phosphorus. We tested a model that AP achieves a portion of this preference by destabilizing ground state binding via charge repulsion between the anionic active site nucleophile, Ser102, and the negatively charged phosphate monoester substrate. Removal of the Ser102 alkoxide by mutation to glycine or alanine increases the observed Pi affinity by orders of magnitude at pH 8.0. To allow precise and quantitative comparisons, the ionic form of bound P(i) was determined from pH dependencies of the binding of Pi and tungstate, a P(i) analog lacking titratable protons over the pH range of 5-11, and from the (3)(1)P chemical shift of bound P(i). The results show that the Pi trianion binds with an exceptionally strong femtomolar affinity in the absence of Ser102, show that its binding is destabilized by >/=10(8)-fold by the Ser102 alkoxide, and provide direct evidence for ground state destabilization. Comparisons of X-ray crystal structures of AP with and without Ser102 reveal the same active site and P(i) binding geometry upon removal of Ser102, suggesting that the destabilization does not result from a major structural rearrangement upon mutation of Ser102. Analogous Pi binding measurements with a protein tyrosine phosphatase suggest the generality of this ground state destabilization mechanism. Our results have uncovered an important contribution of anionic nucleophiles to phosphoryl transfer catalysis via ground state electrostatic destabilization and an enormous capacity of the AP active site for specific and strong recognition of the phosphoryl group in the transition state.
PDB ID: 4KM4Download
MMDB ID: 112081
PDB Deposition Date: 2013/5/8
Updated in MMDB: 2013/08
Experimental Method:
x-ray diffraction
Resolution: 2.8  Å
Source Organism:
Similar Structures:
Biological Unit for 4KM4: dimeric; determined by author and by software (PISA)
Molecular Components in 4KM4
Label Count Molecule
Proteins (2 molecules)
Alkaline Phosphatase(Gene symbol: phoA)
Molecule annotation
Chemicals (8 molecules)
* Click molecule labels to explore molecular sequence information.

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