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Kinetic analysis of SpOatAC and SaOatAC-catalyzed O-acetyltransferase reactions.

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posted on 27.10.2017, 17:38 by David Sychantha, Carys S. Jones, Dustin J. Little, Patrick J. Moynihan, Howard Robinson, Nicola F. Galley, David I. Roper, Christopher G. Dowson, P. Lynne Howell, Anthony J. Clarke

A. pH dependence of the esterase activity catalyzed by SpOatAC (red) and SaOatAC (blue). The specific activities of the enzymes were determined in 20 mM sodium citrate-phosphate-borate buffer at the pH values indicated at 25 oC. B, C. Determination of the steady-state parameters for the esterase activities of SaOatAC and SpOatAC, respectively. Initial velocities of pNP-Ac hydrolysis were determined for the respective enzymes (5 μM) in 50 mM sodium phosphate buffer pH 6.5 containing 5% (v/v) ethanol at 25 oC. D. Determination of the steady state-parameters for the O-acetyltransferase activity of SpOatAC on chitooligosaccharides. The initial velocities of acetyl transfer to the chito-oligosaccharides at the concentrations indicated were determined using 5 μM enzyme in 50 mM sodium phosphate buffer pH 6.5 at 25 oC with pNP-Ac fixed at 2 mM. E. Michaelis-Menten parameters determined for SpOatAC and SaOatAC from the experiments presented in panels B, C and D. All of the enzymatic experiments were performed in triplicate, with the s.e. noted.

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