Models for biological phosphoryl transfer.

Abstract

Quantifying the rate of phosphate monoester dianion hydrolysis under physiological conditions has implications for designing transition state mimics and understanding how catalysis is facilitated. Catalysis is energetically most efficient if the mechanistic pathway in solution is stabilised. Monoesters are believed to have a "dissociative" transition state that has little bonding to the nucleophile and leaving group. However, in many instances, it is suggested that enzymes catalyse monoester transfer through an associative (diester-like) pathway. This 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. By using multiple interactions simultaneously, cooperativity in catalysis may be achieved. However, this 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, the characteristics of alternative (associative) mechanisms that have been proposed for the metallophosphatase catalysed reaction are explored. Finally, complementary catalytic groups are combined with this core complex to look for experimental evidence for possible cooperativity in this context.