Insights into\nthe Mechanism of Paraoxonase-1: Comparing\nthe Reactivity of the Six-Bladed β‑Propeller Hydrolases

The mammalian protein\nparaoxonase-1 (PON1) has been explored\nas\na promising bioscavenger treatment for organophosphorus (OP) agent\npoisoning, but it is not active enough to protect against many agents.\nEngineering is limited because PON1’s catalytic mechanism is\npoorly understood; moreover, its native activity and substrate are\nunknown. PON1 is a calcium-bound six-bladed β-propeller hydrolase\nthat shares high structural homology, a conserved metal-coordinating\nactive site, and substrate specificity overlap with other members\nof a superfamily that includes squid diisopropylfluorophosphatase,\nbacterial drug responsive protein 35, and mammalian senescence marker\nprotein 30. We hypothesized that, by examining the reactivity of all\nfour hydrolases using a common set of conservative mutations, we could\ngain further insight into the catalytic mechanism of PON1. We chose\na set of mutations to examine conserved Asp and Glu residues in the\nhydrolase active sites and the ligation sphere around the catalytic\ncalcium and a His-His dyad seen in PON1. The wild-type (WT) and mutant\nhydrolases were assayed against a set of lactones, aryl esters, and\nOPs that PON1 is known to hydrolyze. Surprisingly, some mutations\nof Ca<sup>2+</sup>-coordinating residues, previously thought to be\nessential for turnover, resulted in significant activity toward all\nsubstrate classes examined. Additionally, merely maintaining WT-like\ncharge in the active site of PON1 was insufficient for high activity.\nFinally, the H115-H134 dyad does not appear to be essential for catalysis\nagainst any substrate. Therefore, previously proposed mechanisms must\nbe re-evaluated.