MurA kinetic assays

his-MurA was purified like his-CwlM. Reactions with 20 µg/ml of his-MurA, equimolar his-CwlM in the kinase reaction, and varying concentrations of substrate were run as in (Brown et al., 1994). MurA kinetic assays were done with MurA_TB alone in MurA reaction buffer (50 mM Tris pH 8.0, 2 mM Kcl, 2 mM DTT) or with equimolar CwlM_TB in a kinase reaction with His-MBP-PknB_KD. The kinase reactions contained: 202 µg/ml CwlM_TB, 50 µg/ml His-MBP-PknB_KD, 2 mM ATP, 2 mM MnCl₂ and were brought up to 100 µl with Buffer C (20 mM Tris pH 7.5, 150 mM NaCl, 1 mM DTT). Inactive kinase reactions had extra buffer instead of ATP. Kinase reactions were run for 1 hr at room temperature before being added to the MurA reactions. MurA reactions contained: 20 µg/ml MurA and 20.2 µg/ml CwlM in the kinase reaction (1/10 of MurA reaction was CwlM kinase reaction, or buffer for MurA alone kinetics). In MurA reactions with varying PEP, UDP-GlcNac was at 10 mM and PEP concentrations were: 25, 100, 250, 500 and 1000 µM; these reactions were started with the addition of UDP-GlcNAc. In MurA reaction with varying UDP-GlcNac, PEP was at 2 mM and UDP GlcNac concentrations were: 0.5, 1, 3, 5, 8, 10 and 15 mM; these reactions were started with the addition of PEP. 2, 5 and 8 min time points were taken for the MurA + CwlM~P reaction; 5, 15 and 25 min time points were taken for the MurA and MurA +CwlM reactions. At the indicated time point, a 25 µl aliquot of the reaction was removed and quenched in an equal volume of 400 mM KOH. The quenched reactions were spun in Microcon 10 K devices (EMD Millipore, Billerica, MA) and chilled until being injected into the Agilent HPLC. HPLC separation was performed on a MonoQ 5/50 GL anion exchange column (GE Healthcare) with the following protocol: 0.6 ml/min; 2 min of 20 mM tetraethylammonium bicarbonate pH 8.0, an 18 min gradient from 20–500 mM tetraethylammonium bicarbonate pH 8.0, 5 min of 500 mM tetraethylammonium bicarbonate pH 8.0, 5 min of 20 mM tetraethylammonium bicarbonate pH 8.0. The area under the A₂₅₄ curve peak corresponding to EP-UDP-GlcNAc was integrated using Agilent ChemStation software.

Because EP-UDP-GlcNac is not commercially available, we were not able to perform a standard curve of product concentrations to determine the relationship between peak area and product concentration. Instead, we measured the peak area of a range of UDP-GlcNac concentrations, and found that a peak area of 11.73 in the A₂₅₄ curve was consistently equivalent to 1 uM of UDP-GlcNac. Because UDP-GlcNac and EP-UDP-GlcNac differ only in one enol-pyruvyl group, we reasoned that the relationship between A₂₅₄ peak area and concentration should be comparable between these two molecules. We therefore divided the values for peak area for each EP-UDP-GlcNac peak by 11.73 to calculate the approximate µM concentration of EP-UDP-GlcNac. These values were plotted vs. time for each substrate concentration and the rate of product produced vs. time was calculated based on the linear portions of the curves. The resulting rates were plotted against substrate concentration in the curves shown in Figure 5 and Figure 5—figure supplement 1. The peak that corresponds to EP-UDP-GlcNAc was identified because it appeared only when MurA and UDP-GlcNAc were incubated with PEP. The data were fitted to the Michaelis Menten formula using GraphPad Prism.