Human Pyruvate Kinase, Muscle

Target ID:

PKM2

Aliases:

CTHBPMGC3932OIP3PK3PKMTCBTHBP1

Deposition Date:

Thursday 28th April 2005

Families:

Non-protein Kinase

Related Diseases:

Metabolism

Structure type:

apo

Download Coordinates:

1ZJH

Authors:

J.CHOE,A.ATANASSOVA,C.ARROWSMITH,A.EDWARDS,M.SUNDSTROM,A.BOCHKAREV,H.PARK,STRUCTURAL GENOMICS CONSORTIUM (SGC)

Site:

Toronto

ICM Datapack:

ICM Datapack

1ZJH

tabs

Structure Details

Pyruvate kinase (PK, EC 2.7.1.40) has been extensively studied
since the mid 1960s, as the enzyme's deficiency in human erythrocytes
is the most common cause of hemolytic anemia.
More than 150 mutations in the gene coding PK in erythrocytes,
designated as RPK, have been identified so far.
The enzyme catalyzes the last step of glycolysis,
where the phosphoryl group of phosphoenolpyruvate (PEP) is transferred to ADP
to form pyruvate and ATP, and thus participates in
the primary intersections of the energy methabolism.
Lately, the enzyme was linked to other diseases related to both glucose and oxygen utilization,
such as diabetes, blood and brain phenylketonuria, and angiogenesis.

The architecture of PK is evolutionally highly conserved
and is organized as a homotetramer with four distinct domains in each subunit.
The activity of the enzyme is a combination of domain and subunits rotations
coupled to active site geometry.
Residues, located in the domain interfaces, play a crucial role in function and communication
between the subunits of the PK. Much progress has been made in the understanding
of the structure and property for RPK, which is expressed in erythrocytes and liver.
Less is known about the two other iso-forms, PKM1 and PKM2,
which are expressed in muscle, kidney and lung,
and are the products of alternative splicing of the same mRNA.

Cations, such as H+, K+, Mg2+ and/or Mn2+,
modulate the activity of PKs.
Mono- or biphosphorylated sugars as well as PEP activate the enzyme and alter its properties.
Phenylalanine is known as a physiological inhibitor of the enzyme.
Indeed, we obtained well diffracting crystals when PKM2 was crystallized
in presence of phenylalanine, Mg2+ and ADP.
However, these ligands and Mg2+ were not located in the structure of PKM2,
suggesting that they acted as additives for the crystallization of PMK2.
The structures of PKM2 in the presence of various activators
and inhibitors are needed to understand the nature of allosteric modulation of the subunits.
These structures will provide the basis for understanding the mechanism of PKM2
activity and addressing the underlying principles of PK-related human diseases.

Materials and Methods

PKM2

PDB:1ZJH

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:GI:33286417
Entry Clone Source:MGC
SGC Clone Accession:
Tag:N-terminal histag with thrombin cleavage site: mgsshhhhhhssglvprgs
Host:

Construct

Prelude:
Sequence:mgsshhhhhhssglvprgsKPHSEAGTAFIQTQQLHAAMADTFLEHMCRLDIDSPPITARNTGIICTIGPASRSVETLKEMIKSGMNVARLNFSHGTHEYHAETIKNVRTATESFASDPILYRPVAVALDTKGPEIRTGLIKGSGTAEVELKKGATLKITLDNAYMEKCDENILWLDYKNICKVVEVGSKIYVDDGLISLQVKQKGADFLVTEVENGGSLGSKKGVNLPGAAVDLPAVSEKDIQDLKFGVEQDVDMVFASFIRKASDVHEVRKVLGEKGKNIKIISKIENHEGVRRFDEILEASDGIMVARGDLGIEIPAEKVFLAQKMMIGRCNRAGKPVICATQMLESMIKKPRPTRAEGSDVANAVLDGADCIMLSGETAKGDYPLEAVRMQHLIAREAEAAIYHLQLFEELRRLAPITSDPTEATAVGAVEASFKCCSGAIIVLTKSGRSAHQVARYRPRAPIIAVTRNPQTARQAHLYRGIFPVLCKDPVQEAWAEDVDLRVNFAMNVGKARGFFKKGDVVIVLTGWRPGSGFTNTMRVVPVP
Vector:p28a-LIC

Growth

Medium:
Antibiotics:
Procedure:We prepared the seeds by inoculating freshly transforming E. coli cells (BL21 DE3) into 80 mL of Luria-Bertani medium. After overnight, all of the seeds were inoculated into 1.8 L of Terrific Broth medium in the presence of 50 Âµg/ml of kanamycin at 37ÂºC and grown to an OD600 of 4.0. Cells were then induced by isopropyl-1-thio-D-galactopyranoside at the final concentration of 1.5 mM and grown overnight at 20ÂºC in a SGC LEX bubbling system.

Purification

Buffers
Procedure
The supernatant was passed through DE52 (Whatman) column equilibrated with the binding buffer and then loaded onto 3 mL Ni-NTA column (Qiagen) equilibrated with the same binding buffer at 4 ºC. The Ni-NTA column was washed with 150 mL of the wash buffer (10mM Tris pH 7.5, 0.5 M NaCl, 5% glycerol, 30 mM imidazole) and the protein was eluted with 15 mL of the elution buffer (10mM Tris pH 7.5, 0.5 M NaCl, 5% glycerol, 250 mM imidazole). The eluate was dialyzed overnight against a buffer containing 10 mM Tris pH 7.5, 0.5 M NaCl, 5% glycerol. The protein concentration was estimated based on the extinction coefficient of the protein, 29190 at 280 nm. Five molar equivalents of ADP, 5 mM TCEP and 5 mM MgCl₂ were added to the purified protein before concentration. The protein was concentrated using an Amicon Ultra centrifugal filter to the final volume of 1 mL and the concentration of 30 mg/mL. About 55 mg of protein was obtained from 1.8 L of cell culture.

Extraction

Buffers
Procedure
Cultures were centrifuged and the cell pellets were suspended in 100 ml of the binding buffer (10 mM Tris pH 7.5, 0.5 M NaCl, 5% glycerol, 5 mM imidazole) with a protease inhibitor cocktail (0.1 mM M benzamidine-HCl and 0.1 mM phenylmethyl sulfonyl fluoride) and flash frozen. The thawed cell pellet was lysed by a combination of 0.5% CHAPS (Sigma) and sonication. The lysate was centrifuged at 15000 rpm for 30 min and the supernatant was used for subsequent steps of purification.
Concentration:
MassSpec:
Crystallization:The supernatant was passed through DE52 (Whatman) column equilibrated with the binding buffer and then loaded onto 3 mL Ni-NTA column (Qiagen) equilibrated with the same binding buffer at 4 ºC. The Ni-NTA column was washed with 150 mL of the wash buffer (10mM Tris pH 7.5, 0.5 M NaCl, 5% glycerol, 30 mM imidazole) and the protein was eluted with 15 mL of the elution buffer (10mM Tris pH 7.5, 0.5 M NaCl, 5% glycerol, 250 mM imidazole). The eluate was dialyzed overnight against a buffer containing 10 mM Tris pH 7.5, 0.5 M NaCl, 5% glycerol. The protein concentration was estimated based on the extinction coefficient of the protein, 29190 at 280 nm. Five molar equivalents of ADP, 5 mM TCEP and 5 mM MgCl₂ were added to the purified protein before concentration. The protein was concentrated using an Amicon Ultra centrifugal filter to the final volume of 1 mL and the concentration of 30 mg/mL. About 55 mg of protein was obtained from 1.8 L of cell culture.
NMR Spectroscopy:
Data Collection:
Data Processing: