High-throughput enzymology reveals mutations throughout a phosphatase that decouple catalysis and transition state analog affinity

Abstract

Using High-Throughput Microfluidic Enzyme Kinetics (HT-MEK), we measured over 9,000 inhibition curves detailing impacts of 1,004 single-site mutations throughout the Alkaline Phosphatase PafA on binding affinity for two transition state analogs (TSAs), vanadate and tungstate. As predicted by catalytic models invoking transition state complementary, mutations to active site and active site-contacting residues had highly similar impacts on catalysis and TSA binding. Unexpectedly, most mutations to more distal residues which reduced catalysis had little or no impact on TSA binding and many even increased affinity for tungstate. These disparate effects are accounted for by a model in which distal mutations alter the enzyme's conformational landscape and increase occupancy of microstates that are catalytically less effective but better able to accommodate larger transition state analogs. In support of this model, glycine substitutions (rather than valine) were more likely to increase tungstate affinity, presumably due to increased conformational flexibility and increased occupancy of previously disfavored microstates. These results indicate that residues throughout an enzyme provide specificity for the transition state and discriminate against analogs that are larger only by tenths of an Ångström. Thus, engineering enzymes that rival the most powerful natural enzymes will likely require consideration not just of residues in and around the active site, but also of more distal residues that shape the enzyme's conformational landscape and fine-tune the active site. In addition, the extensive functional communication between the active site and remote residues may provide interconnections needed for allostery and make allostery a highly evolvable trait.

Second-Shell Residues Contribute to Catalysis by Predominately Preorganizing the Apo State in PafA

“…For D163V, the measured decrease in k cat /K M with MeP (all experimental data were reported as base-2 logarithm values for the ratio of WT and mutant properties 40,44 ) is −3.1, which is close to the increase of 3.1 in the K d of P i ; the increase of tungstate K d is slightly lower, 2.3. 44 By comparison, for Y112V, the measured decrease in k cat /K M with MeP is −3.7, the increase in K d of P i is 2.9, and the increase in K d of tungstate is 4.4.…”

“…The values in the parentheses for Δ G ‡ and Δ G are the corresponding statistical uncertainties (see Materials and Methods for details). b The experimental K i values are for inorganic phosphate c Dissociation constants for two transition state analogues (TSAs); the values without parentheses are for vanadate, and those with parentheses are for tungstate. d YD stands for the Y112V/D163V double mutant. …”

“…b The experimental K i values are for inorganic phosphate. 40 c Dissociation constants for two transition state analogues (TSAs); 44 the values without parentheses are for vanadate, and those with parentheses are for tungstate. d YD stands for the Y112V/D163V double mutant.…”

“…Second-shell residues may also control other important characteristics of the active site, such as electrostatics , and the level of hydration. Since transition state stabilization is a fundamental feature of enzymes, it is often assumed that the positioning effect of second-shell residues is manifested in the transition state . However, considering the fact that the catalytic properties of enzymes involve multiple functional states, it remains unclear in which functional state(s) the positioning effect of second-shell residues is most impactful.…”

“…While the largest impact was from mutations of first-shell residues (Figure 1b), it was observed that many second-shell residues and beyond make considerable contributions to catalytic efficiency (k cat /K M ), substrate specificity (the ratio of phosphate monoester and diester activities), product inhibition (inorganic phosphate binding), and the binding affinity of the transition state analogues. 44 For example, mutations of residues outside the active site were observed to cause a notable degree of decrease in k cat /K M for methyl phosphate monoester (MeP) hydrolysis, highlighting that the extraordinary catalytic efficiency of PafA is not merely determined by the bimetallic core and the first-shell residues. The availability of the systematic kinetics data set, together with ample structural and biochemical information on PafA, 41,42 makes it an ideal system for better elucidating the roles of residues beyond the first coordination shell in enzymes.…”