enzymology presentation 1260077

11
Enzymology presentation By Titas Mandal IMTH-VI 1260077 Guided by Dr. Rahul Modak

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Page 1: Enzymology presentation 1260077

Enzymology presentation

By Titas MandalIMTH-VI1260077

Guided by Dr. Rahul Modak

Page 2: Enzymology presentation 1260077
Page 3: Enzymology presentation 1260077

• MetN1 methionine transporter is an ATP binding Cassette (ABC) transporter in E.Coli

• Energy dependent uptake of D & L-Methionine against i.e. against the gradient-ATP dependent

• Uptake of Methionine is governed by the internal methionine pool of the cell Transinhibition

TMD: Transmembrane domainNBD: Nucleotide binding domain

Page 4: Enzymology presentation 1260077

The ExperimentProtein purification and extraction:• Wild type His-tagged MetN1 expression plasmid transformed into BL21-Gold cells.

• Cells cultured with IPTG to induce protein expression.

• Cells harvested by centrifugation and stored at -80°C.

• Homogenization of frozen cell pellets in purification buffer aided with Dnase, lysozyme and protease inhibitors

• DDM (n-dodecyl-β-D-maltopyranoside) added for MetN1 extraction. DDM is the detergent.

• Use of microfluidizer for more efficient and further homogenization and centrifugation to obtain the supernatant.

• HisTrap FF columns for purification of His tagged recombinant proteins and Imidazole during elution for their

extraction.

• Extract passed through Superdex 200 column and concentrated in 100 kDa MW cutoff centrifuge filter.

Page 5: Enzymology presentation 1260077

ATPase assays :

• Amount of inorganic phosphates measured by Enzchek phosphate assay kit.

• Reaction mixture contained MgCl2 and ATP along with L-Methionine.

• Reaction initiated by automatic injection of purified MetN1 into the mixture.

Kinetics of ATP hydrolysis was examined to elucidate the mechanism of MetN1 transinhibition.

For measurement of KI (ADP):

• Initial rates were obtained by calculating the linear portion of the graph of ΔA at 310 nm wrt time using

Megallen software.

• Initial rates were plotted against inhibitor concentration.

• Fitted to the following equation.

Page 6: Enzymology presentation 1260077

For determination of Km (ATP), Vmax and Hill coefficient nATP:

• Initial reaction rate was plotted against the ATP concentration.

• Fitted to the following equation

Page 7: Enzymology presentation 1260077

Isothermal titration calorimetry:

• Further concentration of MetN1 using Amicon ultra 100 Kda MW cutoff centrifugal filter that provides great

performance and lower spin time.

• Overnight dialysis and recentrifugation for clearing.

• MetN1 titrated with L-Met using MicroCal iTC 200. Highly sensitive low volume titration calorimeter.

Page 8: Enzymology presentation 1260077

Results:1.Under subsaturating ATP concentrations, ADP inhibited MetNI ATPase activity with a KI(ADP) of 41 ± 2 μM and a Hill coefficient of 1.3 ± 0.1, indicating low cooperativity for the binding of ADP.

ATP conc. was varied under constant ADP conc. both above and below the measured Ki (ADP)In the presence of ADP the apparent Vmax remained constant while the apparent Km(ATP)

increased with increasing inhibitor conc.

Experiment confirmed Competitive inhibition by ADP

Page 9: Enzymology presentation 1260077

2.L-Met inhibition of MetN1Inhibition experiments under saturating ATP concentrations were fit to a KI(L-Met) of 41 ± 1 μM and a Hill coefficient of 1.4 ± 0.1, indicating that there is more than one methionine binding site per MetNI transporter and low cooperativity between the sites

With increasing L-Met conc. the Vmax decreased with increasing meth conc. with apparently constant Km(ATP)

L-methionine is a non-competitive inhibitor for

ATPase activity of MetN1

Page 10: Enzymology presentation 1260077

Discussions:

L-Met can bind to the MetN1 at allosteric sites resulting in non- competitive inhibition of ATP

hydrolysis.

De novo biosynthesis of meth is energetically expensive(7 ATPs) as compared to its import that

costs only 2 ATPs.

Homeostatic intracellular concentration of Met is maintained by a balance between its

disappearance by protein synthesis and appearance by import or biosynthesis.

Intracellular Met concentration is inversely proportional to the transport rate by MetN1.

Page 11: Enzymology presentation 1260077

Thank you