Cooperative Electrostatic Interactions Drive Functional Evolution in the Alkaline Phosphatase Superfamily

Abstract

It is becoming widely accepted that catalytic promiscuity, i.e., the ability of a single enzyme to catalyze the turnover of multiple, chemically distinct substrates, plays a key role in the evolution of new enzyme functions. In this context, the members of the alkaline phosphatase superfamily have been extensively studied as model systems in order to understand the phenomenon of enzyme multifunctionality. In the present work, we model the selectivity of two multiply promiscuous members of this superfamily, namely the phosphonate monoester hydrolases from Burkholderia caryophylli and Rhizobium leguminosarum. We have performed extensive simulations of the enzymatic reaction of both wild-type enzymes and several experimentally characterized mutants. Our computational models are in agreement with key experimental observables, such as the observed activities of the wild-type enzymes, qualitative interpretations of experimental pH-rate profiles, and activity trends among several active site mutants. In all cases the substrates of interest bind to the enzyme in similar conformations, with largely unperturbed transition states from their corresponding analogues in aqueous solution. Examination of transition-state geometries and the contribution of individual residues to the calculated activation barriers suggest that the broad promiscuity of these enzymes arises from cooperative electrostatic interactions in the active site, allowing each enzyme to adapt to the electrostatic needs of different substrates. By comparing the structural and electrostatic features of several alkaline phosphatases, we suggest that this phenomenon is a generalized feature driving selectivity and promiscuity within this superfamily and can be in turn used for artificial enzyme design.

Leaving Group Ability Observably Affects Transition State Structure in a Single Enzyme Active Site

“…4,5 We have observed that rather modest changes in the reactivity of a substrate can have significant, conspicuously observable effects on the nature of a TS without changing the chemical mechanism or rate-determining step. The fact that AP catalyzes the hydrolysis of the whole range of LGs largely bolsters previous notions about catalytic promiscuity in AP 9,28,58,59 and similar systems, 60,61 namely that the enzyme is capable of stabilizing TSs with a variety of structures and that it does so by different means. Nonetheless, our results indicate significant departures from the previously proposed reaction pathways taken by AP’s phosphate ester substrates.…”

Perspective: Defining and quantifying the role of dynamics in enzyme catalysis

“…Thus, it was not until the 1976 work of Warshel and Levitt 51 that the major role of electrostatic effects in catalysis was clearly demonstrated. However, since 1976, it has become quite clear that electrostatic effects are central to catalysis 1 (and more recently also to enzyme functional evolution 157 ). The nature of the electrostatic stabilization has been recognized first in Ref.…”

Structural plasticity mediates distinct GAP‐dependent GTP hydrolysis mechanisms in Rab33 and Rab5

“…For example, enzymes catalyzing phosphoryl transfer reactions are known to have backup active site residues for the catalysis of promiscuous reactions [29,30]. For example, enzymes catalyzing phosphoryl transfer reactions are known to have backup active site residues for the catalysis of promiscuous reactions [29,30].…”

“…Structural plasticity is a general concept governing the function of many proteins. For example, enzymes catalyzing phosphoryl transfer reactions are known to have backup active site residues for the catalysis of promiscuous reactions [29,30]. In case of GTPases, structural plasticity is even more critical to ensure versatility in effector binding and consequently the effective activation/inactivation of GTPases.…”