Kinetics of glycosyl hydrolase family 2 beta-glucuronidases
September 15-19, 2019
Glucuronidases serve important roles in physiology, research, and commercial applications. Bacteria of the microbiome produce β-glucuronidase (β-GUS) to scavenge glucuronic acid from compounds excreted via phase II metabolism. In research, β-GUS is commonly used as a reporter for gene expression. Commercially, β-GUS is used in clinical and forensic drug testing to cleave glucuronic acid from drug molecules making them easier to quantify by LC-MS. Minimally, the enzyme must form a homodimer for activity, and the naturally occurring active form is believed to be a tetramer. Past studies have investigated hydrolysis activity of the enzyme and modeled it as simple Michaelis-Menten kinetics. In our search for new enzymes and enzyme variants, we discovered a point mutation that radically alters the kinetics of the enzyme and points to a strong allosteric interaction between enzyme subunits. We further observe substrate inhibition and consider inhibition by competing substances found in clinical samples. Here we survey several different enzymes and variants across multiple substrates and present models for evaluating enzyme performance.
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John J. Tomashek, Caleb R. Schlachter, Gary C. Horvath, Nanda G. Karri, and L. Andrew Lee, "Kinetics of glycosyl hydrolase family 2 beta-glucuronidases" in "Enzyme Engineering XXV", Huimin Zhao, University of Illinois at Urbana-Champaign, USA John Wong, Pfizer, USA Eds, ECI Symposium Series, (2019). https://dc.engconfintl.org/enzyme_xxv/60