Abstract 1926 Kinetics of Pyrophosphate Release Catalyzed by Nudix Hydrolase Enzymes

Nudix proteins comprise a superfamily of enzymes that hydrolyze nucleoside diphosphates linked to another moiety (X). The first nudix proteins characterized are homologs of the E. coli MutT protein, which cleaves pyrophosphate from (deoxy)nucleoside triphosphates ((d)NTPs) to prevent nucleotide misincorporation during DNA replication and subsequent mutations. MutT-like nudix proteins are widely believed to preferentially hydrolyze oxidatively damaged dNTPs, like 8-oxo-dGTP to prevent G:A mismatches, which lead to A:T to C:G transversion mutations. Evidence for this preference stems mainly from dramatically low Km values for mutagenetic NTPs observed in steady-state reactions where release of pyrophosphate is indirectly observed using HPLC, colorimetric reagents, or radiolabeled phosphates. Here, the kinetics of nudix-catalyzed pyrophosphate release were more directly observed by coupling the reaction to ATP production using sulfurylase and adenosine-5'-phosphosulfate. ATP production could then be monitored in real time either by using firefly luciferase or by using hexokinase, glucose, NADP+, and glucose-6-phosphate dehydrogenase. Both assays were optimized and used to characterize the substrate specificity of a variety of purified nudix enzymes from humans, E. coli, and pathogenic bacteria. Both assays could be useful in high-throughput screening campaigns designed to identify inhibitors of nudix proteins, especially those linked to cancer. Supported by NIH grant 1R15GM144859.