Models for biological phosphoryl transfer

Abstract

Phosphoryl transfer at the phosphate mono-and diester level is ubiquitous in biology. However, the detailed understanding of how these reactions proceed and the corresponding enzyme catalysed processes are still the subject of debate despite the extensive physical organic chemistry data that has accumulated over the years. Recent work has revealed that both processes are extremely slow under physiological conditions, and that the corresponding efficiencies of the enzymes that catalyse these reactions are the highest established to date [1,2]. This suggests that these enzymes have very high transition state affinities, and so will be very sensitive to accurate transition state mimics. Despite the observation that alkyl phosphate mono-and diesters have comparable reactivity, the transition states for hydrolysis in solution are significantly different. Diesters are believed to react through a phosphorane intermediate or phosphorane-like ("associative") transition state. Monoesters are believed to have a "dissociative" transition state that has much less bonding to the nucleophile and leaving group. This obviously has implications for transition state mimics and the understanding of how catalysis is facilitated. This is energetically most efficient if the mechanistic pathway in solution is stabilised; however, in many instances, it is suggested that enzymes catalyse monoester transfer through an associative (diester-like) pathway. This means that the rate acceleration has to be yet higher to account for the observed rates, but is perhaps easier to rationalise in terms of the active site residues available. For example, in the catalytic subunit of protein phosphatase 1 (PP1), these are metal ions and cationic side chains which might be expected to stabilise developing negative charge (see figure below left). This leads on to the idea that by using multiple interactions simultaneously, cooperativity in catalysis is achieved i.e. the overall effect is greater than the sum of the parts. However, this intriguing idea is difficult to demonstrate unambiguously in large, complex natural systems. This contribution examines the background reactivity of phosphate esters, and reports data showing that the substrates for serine/threonine phosphatases have slower intrinsic rate constants than any other enzyme substrates. Using model complexes (see figure above right), the characteristics of alternative (associative) mechanisms that have been proposed for the metallophosphatase catalysed reaction are explored [3,4]. Finally, complementary catalytic groups are combined with this core complex to look for experimental evidence for possible cooperativity in this context [5,6].

A Direct Substrate–Substrate Interaction Found in the Kinase Domain of the Bifunctional Enzyme, 6-Phosphofructo-2-kinase/Fructose-2,6-bisphosphatase

“…The distance between the phosphoryl acceptor and the γ-phosphorus, and the geometric arrangement of the three atoms together suggest an "in-line" transfer of phosphate accompanied with inversion of phosphorus configuration. 25,26 This structural observation coincides with the previous biophysical observation on the stereochemical course of phosphoryl transfer catalyzed by PFKFB1. 22 When the configuration of transferred phosphorus atom was tested by 31 P NMR spectroscopy, using [γ-(S)- 16 O, 17 O, 18 O]ATP as probing substrate, an in-line transfer with a net inversion of configuration was observed.…”

Structural Insights into the Catalytic Mechanism of Phosphate Ester Hydrolysis by dUTPase

“…The Character of Phosphate Ester Hydrolysis-Available data for non-enzymatic phosphate diester hydrolysis reactions are in favor of a mechanism with significant associative character, since the metaphosphate transition state required for the dissociative mechanism is highly unstable when an additional bulky ligand is present (45,46). Our present results for the dUTPase-catalyzed reaction are in agreement with this expectation.…”

Theoretical Evaluation of the Substrate‐Assisted Catalysis Mechanism for the Hydrolysis of Phosphate Monoester Dianions

“…Physical organic chemists have traditionally assumed that a methyl group is a valid substitute for a phosphate hydrogen atom and have accordingly often speculated, in order to reject the SAC alternative, that the rate constant for OH À attack on ROPO 3 H À should be roughly the same as that for OH À attack on the corresponding methylated diester monoanion. [15,19] Whether or not this assumption is justified is an important question that can only be resolved by careful quantum studies. However, when reactions in aqueous solution are considered and when charged reactants with hydrogen-bond-donating and -accepting ability, such as OH À and ROPO 3 H À , are involved, it seems reasonable to assume that quantum approaches will not be a faithful comparison to experiment unless the calculations include an appropriate (large) number of explicit solvent molecules and extensive dynamics.…”