The use of reaction timecourses to determine the level of minor contaminants in enzyme preparations

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• 作者：Lawrence M. Goldman ; ¹ ; ^{goldmalm@whitman.edu" class="auth_mail} ; Tina L. Amyes
• 关键词：Enzyme purity ; Enzyme kinetics
• 刊名：Analytical Biochemistry
• 出版年：1 April, 2014
• 年：2014
• 卷：450
• 期：Complete
• 页码：20-26
• 全文大小：809 K

文摘

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鈥?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 0.093 mol%. 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.