Talk Abstracts

Talk on Wednesday 03:30-03:45pm submitted by Timothy Grunkemeyer

Rational and combinatorial exploration of the catalytic mechanism of Paraoxonase-1

Timothy J. Grunkemeyer (Ohio State Biochemistry Program), Kiran Doddapaneni (Department of Chemistry and Biochemistry)

Abstract:
Each year over 3 million people are exposed to toxic organophosphorus compounds (OPs), which target the parasympathetic nervous system. Since the current treatment is only moderately effective, we aim to generate a novel therapeutic in the form of a catalytic bioscavenger. Paraoxonase-1 (PON1) has shown promise as a new drug for OP treatment however, its mechanism is poorly understood, thus limiting our ability to engineer PON1 for improved therapeutic efficacy. To understand its mechanism, we aim to interrogate key catalytic residues using rational and combinatorial methods. The primary residues of interest are an Asp and a His-dyad in the base and side of the active site respectively. It has been hypothesized that any of these residues can activate a water for substrate hydrolysis or attack the substrate directly. To test this, we cloned, expressed, and purified a set of rational mutations in PON1 that were screened against aryl-esters, lactones, and OPs. k_cat/K_m values were calculated to determine the effect on catalysis. In Asp to Asn/Glu mutations, only moderate losses in activity are observed while His to Phe mutants result in a 4x-8x increase in both K_m and k_cat. Mutation of the Asp and His-dyad together result in only a two order of magnitude loss in activity. These observations argue against the His-dyad participating in catalysis, but indicate a potential role in substrate binding while indicating the importance of Asp as a metal binding residue. To confirm this, a library that randomized the His-dyad was cloned and screened. Analysis of the hits identified from this library and subsequent rational variants indicate the identity of these residues may not be critical, but specific biophysical properties are essential for proper substrate binding and orientation for productive attack by a side chain or water. Furthermore, it can be noted that the specificity of the enzyme can be tuned by modulating the characteristics of the dyad partners.

Keywords: hydrolysis, enzyme, metal