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Metformin is one of the most regularly prescribed Type II diabetes drugs in the world, and its use is likely to expand as diabetes diagnoses rise globally. This drug and its main degradation byproduct, guanylurea, are not fully metabolized by humans and cannot be removed through conventional water treatment processes. These compounds have been detected in coastal waters around the world and are currently considered emerging pollutants. The goal of this research was to examine the catalytic mechanism and substrate specificity of Guanylurea Hydrolase (GuuH), a recently discovered enzyme that converts guanylurea to ammonia and guanidine. Bioinformatic analyses were conducted to predict the active site and three-dimensional structure of GuuH. Site-directed mutagenesis was performed to construct mutants in amino acids predicted to be part of the enzyme's catalytic triad and substrate binding site. The mutants created were K138R, N141K, E211D, E211Q, and E211N. The wild-type and mutant enzymes were purified using His-tag affinity chromatography. Enzyme activity was assessed by measuring ammonia released using Berthelot assays. The results showed that the K138R mutant had similar specific activity compared to the wild-type GuuH when reacting with guanylurea, while E211N and E221D showed low specific activity under the same conditions. All of the enzymes had no detectable activity when reacting with biuret, which suggests they have low affinity for this substrate. Future work will focus on kinetic analyses of the wild-type and K138R enzymes and additional mutagenesis to identify the amino acids that determine the substrate specificity to the enzyme. Understanding GuuH's catalytic activity and substrate specificity is essential to using this enzyme in the development of biotechnological applications for water treatment.