MurA kinetic assays

Final MurA enzyme kinetics protocol

Vary UDP-GlcNAc concentrations

Set up one set of reactions with varying UDP-GlcNAc concentrations. Take 5, 15, 25 min time points for each.

Reaction Buffer (RxB): 50 mM Tris pH 8.0, 2 mM KCl, 2 mM DTT. Buffer C: 20 mM Tris pH 7.5, 150 mM NaCl, 1 mM DTT.

PEP stock is at 100 mM. Dilute 1/5 in RxB to make a 20 mM stock.

Mixed 3.3 mg of UDP-GlcNac into 50 µl 10 mM Tris pH8 to make a 100 mM stock. Dilute 1/10 for a 10 mM stock.

Concentrations of UDP-GlcNac are 500, 1000, 3000, 5000, 8000, 10,000, 12,000, 15,000 uM . Use PEP at 2 mM. 

Use MurA at 20 ug/ml=  1.6 ug/ 80 ul MurA reaction. Equimolar CwlM is 20.2 ug/ml or 1.602 ug/ 80 ul MurA reaction.

Make 80 µl reactions for each substrate concentration. Take 22 µl aliquots at each time point and quench with 22 µl 0.4M KOH at the indicated time. Then spin through a microcon 10K and ice until HPLC.

100 ul CwlM kinase reaction

HCwlM is at 0.45 mg/ml. Mix and incubate at RT for 1 hour:

45 ul HCwlM

2 ul 3 mg/ml PknB

8 ul MnCl2

45 ul buffer C

In this reaction HCwlM is at 202 ug/ml. Use a 1/10 dilution of this in each MurA reaction 

Varying UDP-GlcNac concentrations

1. MurA is at 0.150 ug/ul. Use 10.7 ul in each 80 ul reaction for 1.6 ug. HCwlM in the kinase reaction is at 0.202 ug/ ul, use 8 ul of the kinase reaction for each MurA reaction.
2. First put buffer, UDP-GlcNAc and MurA in a PCR strip. Put 25 µl KOH in rows of a PCR plate. 
3. Start reactions by adding PEP at the same time with a multichannel.
4. Quench 25 ul of reaction into the PCR plate at each time point. Keep the plate on ice, keep the reaction tubes at 37°
5. After quenching, spin each reaction through a microcon 10K filter device, take filtrate and run on HPLC 

For each 80 ul reaction add:

• 10.7 ul 0.15 ug/ul HMurA
• 8 ul HCwlM kinase reaction

1. 0.5 mM UDP-GlcNac
   1. 4 µl 10 mM UDP-GlcNac
   2. 49.3 µl RxB
2. 1 mM UDP-GlcNac
   1. 8 ul 10 mM UDP-GlcNac
   2. 45.3 ul RxB
3. 3 mM UDP-GlcNac
   1. 24 ul 10 mM UDP-GlcNac
   2. 29.3 ul RxB
4. 5 mM UDP-GlcNac
   1. 40 ul 10 mM UDP-GlcNac
   2. 13.3 µl RxB
5. 8 mM UDP-GlcNAc
   1. 6.4 ul 100 mM UDP-GlcNAc
   2. 46.9 ul RxB
6. 10 mM UDP-GlcNAc
   1. 8 ul 100 mM UDP-GlcNac
   2. 45.3 ul RxB
7. 12 mM UDP-GlcNac
   1. 9.6 ul 100 mM UDP-GlcNac
   2. 43.7 ul RxB
8. 15 mM UDP-GlcNac
   1. 12 ul 100 mM UDP-GcNac
   2. 41.3 ul RxB

Then add 8 ul of 20 mM PEP (final concentration = 2mM) to all reactions at once and start timer, start taking time points.

Vary PEP concentrations

Set up one set of reactions with varying PEP concentrations. Take 2, 5, 8 min time points for each for CwlM~P reaction,  and 5, 15, 25 min time points for others

Reaction Buffer (RxB): 50 mM Tris pH 8.0, 2 mM KCl, 2 mM DTT. Buffer C: 20 mM Tris pH 7.5, 150 mM NaCl, 1 mM DTT.

PEP stock is at 100 mM. Dilute 1/10 in RxB to make a 10 mM stock.

Mixed 3.3 mg of UDP-GlcNac into 50 µl 10 mM Tris pH8 to make a 100 mM stock. 

Saturating concentration of UDP-GlcNac is 10 mM. Use PEP at 0.025, 0.1, 0.25, 0.5 and 1 mM. 

Use MurA at 20 ug/ml=  3 ug/ 300 ul MurA reaction (46326 MW) = 0.432 nM

Use HCwlM at 20.2 ug/ml (46723) MW = 0.432 nM

MurA reactions should have 20.2 ug/ ml of HCwlM, so kinase reaction should have 10X this much – 202 ug/ml

Make 150 µl reactions for each substrate concentration. Take 25 µl aliquots at each time point and quench with 25 µl 0.4M KOH at the indicated time. Then spin through a microcon 10K and ice until HPLC.

100 ul CwlM kinase reaction

HCwlM is at 0.51 mg/ml. Mix and incubate at RT for 1 hour:

40 ul HCwlM

2 ul 3 mg/ml PknB

8 ul MnCl2

8 ul ATP

42 ul buffer C

In this reaction HCwlM is at 202 ug/ml = 0.202 ug/ul = 20.2 ug in the kinase reaction total, and 20.2 ug/ml of CwlM in the MurA reactions, i.e., a 1/10 dilution

Varying PEP concentrations

6. MurA is at 0.150 ug/ul. Use 12 ul in each 90 ul reaction for 3 ug. HCwlM in the kinase reaction is at 0.202 ug/ ul, use 9 ul of the kinase reaction for each MurA reaction.

7. First put buffer, PEP and MurA in a PCR strip. Put 25 µl KOH in rows D-H of a PCR plate. 

8. Start reactions by adding UDP-GlcNac at the same time with a multichannel.

9. Quench 25 ul of reaction into the PCR plate at each time point. Keep the plate on ice, keep the reaction tubes at 37°

10. After quenching, spin each reaction through a microcon 10K filter device, take filtrate and run on HPLC 

For each 150 ul MurA reaction add:

• 12 ul 0.15 ug/ul HMurA
• 9 ul HCwlM kinase reaction

9. 0.025 mM PEP

     a. 2.25 µl 1 mM PEP

     b. 57.75 µl RxB

10. 0.1 mM PEP

     a. 9 µl 1 mM PEP

     b. 51 µl RxB

11. 0.25 mM PEP

     a. 22.5 µl 1 mM PEP

     b. 37.5 µl RxB

12. 0.5 mM PEP

     a. 4.5 µl 10 mM PEP

     b. 55.5 µl RxB
13. 1 mM PEP

     a. 9 ul 10 mM PEP

     b. 51 ul RxB

Then add 9 ul of 100 mM UDP-GlcNac to all reactions at once and start timer, start taking time points.

Sample prep and HPLC

Samples were each spun at 4° in a Microcon 10K filtration device (EMD Millipore, Billerica, MA) to remove the proteins, and the flow-through was kept at 4° until running on the 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. The concentration of EP-UDP-GlcNac, the product, was calculated by assuming that it absorbs the same as UDP-GlcNac at A254. A standard curve of UDP-GlcNAc was run through the HPLC protocol to determine the relationship between concentration and area under the curve – the same conversion factor was used for EP-UDP-GlcNac.

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