title: The use of reaction timecourses to determine the level of minor contaminants in enzyme preparations.
abstract: Enzyme mutagenesis is a commonly used tool to investigate the structure and activity of enzymes. However, even minute contamination of a weakly active mutant enzyme by a considerably more active wild-type enzyme can partially or completely obscure the activity of the mutant enzyme. In this work, we propose a theoretical approach using reaction timecourses and initial velocity measurements to determine the actual contamination level of an undesired wild-type enzyme. To test this method, we applied it to a batch of the Q215A/R235A double mutant of orotidine 5'-monophosphate decarboxylase (OMPDC) from Saccharomyces cerevisiae that was inadvertently contaminated by the more active wild-type OMPDC from Escherichia coli. The enzyme preparation showed significant deviations from the expected kinetic behavior at contamination levels as low as 0.093mol%. We then confirmed the origin of the unexpected kinetic behavior by deliberately contaminating a sample of the mutant OMPDC from yeast that was known to be pure, with 0.015% wild-type OMPDC from E. coli and reproducing the same hybrid kinetic behavior.
authors.affiliation: Department of Chemistry, University at Buffalo, Buffalo, NY 14260, USA. Electronic address: goldmalm@whitman.edu.
authors.forename: Lawrence M
authors.initials: LM
authors.lastname: Goldman
authors.affiliation: Department of Chemistry, University at Buffalo, Buffalo, NY 14260, USA.
authors.forename: Tina L
authors.initials: TL
authors.lastname: Amyes
mesh_terms: D055162:Biocatalysis; D004340:Drug Contamination; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D009154:Mutation; D009964:Orotidine-5'-Phosphate Decarboxylase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology
chemical_list: D009964:Orotidine-5'-Phosphate Decarboxylase
keywords: Enzyme kinetics; Enzyme purity