Extensive site-directed mutagenesis reveals interconnected functional units in the alkaline phosphatase active site

Abstract

Enzymes enable life by accelerating reaction rates to biological timescales. Conventional studies have focused on identifying the residues that have a direct involvement in an enzymatic reaction, but these so-called ‘catalytic residues’ are embedded in extensive interaction networks. Although fundamental to our understanding of enzyme function, evolution, and engineering, the properties of these networks have yet to be quantitatively and systematically explored. We dissected an interaction network of five residues in the active site of Escherichia coli alkaline phosphatase. Analysis of the complex catalytic interdependence of specific residues identified three energetically independent but structurally interconnected functional units with distinct modes of cooperativity. From an evolutionary perspective, this network is orders of magnitude more probable to arise than a fully cooperative network. From a functional perspective, new catalytic insights emerge. Further, such comprehensive energetic characterization will be necessary to benchmark the algorithms required to rationally engineer highly efficient enzymes.DOI: http://dx.doi.org/10.7554/eLife.06181.001

Tungstate as a Transition State Analog for Catalysis by Alkaline Phosphatase

“…S1a and b). Whereas prior work has demonstrated that the metal content of AP in solution is two Zn 2+ ions and one Mg 2+ ion and per active site [4,18], substitution at the Mg 2+ site has previously been observed crystallographically [19]. The comparison of two previously obtained sub-2 Å resolution structures of phosphate-bound AP, one with full Mg 2+ occupancy (3TG0 from Ref.…”

“…However, the Mg 2+ ion and its associated water network provide significantly more transition state-specific stabilization compared to Arg166, which stabilizes both the transition and ground states (Fig. 1b) [4,5,18]. The relative contributions of the Mg 2+ ion to binding affinity versus rate enhancement would thus be expected to probe how well a ligand mimics the energetics specific to AP’s transition state, so the insensitivity of vanadate but not tungstate binding affinity to loss of the Mg 2+ ion is particularly striking in the context of transition state analog behavior (Fig.…”

“…The chemical step is, however, rate-limiting for WT hydrolysis of the less reactive substrate methyl phosphate [3], so the WT kcat/KM value listed is the kcat/KM,obs value measured for methyl phosphate multiplied by the ratio of the kcat/KM values observed for pNPP and methyl phosphate for AP mutants for which the chemical step is rate-limiting. This ratio of 20 is constant across several mutants, supporting its use as a correction factor to estimate the expected kcat/KM value [18]. …”

“…1c. Errors are noted in parenthesis and correspond to the standard deviations from 2–3 independent experiments. b Values are reproduced from Sunden et al [18], in which rates were measured by monitoring the hydrolysis of p-nitrophenol phosphate (pNPP) for all AP variants except WT, because the chemical step is not rate-limiting for WT AP with this substrate [3]. The chemical step is, however, rate-limiting for WT hydrolysis of the less reactive substrate methyl phosphate [3], so the WT kcat/KM value listed is the kcat/KM,obs value measured for methyl phosphate multiplied by the ratio of the kcat/KM values observed for pNPP and methyl phosphate for AP mutants for which the chemical step is rate-limiting.…”

Mechanistic and Evolutionary Insights from Comparative Enzymology of Phosphomonoesterases and Phosphodiesterases across the Alkaline Phosphatase Superfamily

“…(B) k cat / K M values for phosphomonoester (Me-P) hydrolysis by WT PafA (black) and Ec AP (gray). Values are from Table 3 and ref (17). …”