Poster abstracts
Poster number 65 submitted by Timothy Grunkemeyer
New insights into a novel Paraoxonase-1 catalytic mechanism
Timothy Grunkemeyer (OSBP), Kiran Doddapaneni (Department of Chemistry and Biochemistry), Srividya Murali (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, resulting in death. 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, due to a plethora of conflicting evidence, its mechanism is poorly understood limiting our abilities to engineer PON1 and ultimately its therapeutic efficacy. To understand PON1’s mechanism, we aim to interrogate its active site structurally and kinetically. The primary residues of interest are an Asp and His in the base and side of the active site respectively. It has been hypothesized that either of these residues can activate a water for substrate hydrolysis or attack the substrate directly forming an enzyme-substrate complex that is subsequently hydrolyzed. We hypothesize that the enzyme operates via hydrolysis from a side chain activated water. To test these hypotheses, we cloned, expressed, and purified a set of conservative mutations in PON1. Subsequently, they were kinetically screened against a panel of aryl esters, lactones, and OPs. kcat/Km values were calculated to determine the effect on catalysis. In Asp to Asn/Glu mutation, we observe only a moderate loss in activity while the His to Phe mutants result in a 4x-8x increase in both Km and kcat. Interestingly, mutation of both the Asp and His result in only a two order of magnitude loss in activity. These observations argue against the His participating in catalysis, but indicate a potential role in substrate binding. The above results support a mechanism in which water is non-specifically activated by any general base in the active site creating a pool of OH- ions that can be used for hydrolysis. Crystallographic analyses of notable mutants and chiral substrate hydrolysis are under way to decipher the aspartate’s exact role in catalysis.
Keywords: Enzyme, Hydrolysis, Mechanism