Differential Behavior of Missense Mutations in the Intersubunit Contact Domain of the Human Pyruvate Kinase M2 Isozyme

Abstract

In this study, we attempted to understand the mechanism of regulation of the activity and allosteric behavior of the pyruvate kinase M 2 enzyme and two of its missense mutations, H391Y and K422R, found in cells from Bloom syndrome patients, prone to develop cancer. Results show that despite the presence of mutations in the intersubunit contact domain, the K422R and H391Y mutant proteins maintained their homotetrameric structure, similar to the wild-type protein, but showed a loss of activity of 75 and 20%, respectively. Interestingly, H391Y showed a 6-fold increase in affinity for its substrate phosphoenolpyruvate and behaved like a non-allosteric protein with compromised cooperative binding. However, the affinity for phosphoenolpyruvate was lost significantly in K422R. Unlike K422R, H391Y showed enhanced thermal stability, stability over a range of pH values, a lesser effect of the allosteric inhibitor Phe, and resistance toward structural alteration upon binding of the activator (fructose 1,6-bisphosphate) and inhibitor (Phe). Both mutants showed a slight shift in the pH optimum from 7.4 to 7.0. Although this study signifies the importance of conserved amino acid residues in long-range communications between the subunits of multimeric proteins, the altered behavior of mutants is suggestive of their probable role in tumor-promoting growth and metabolism in Bloom syndrome patients with defective pyruvate kinase M 2 .Pyruvate kinase (PK 3 ; EC 2.7.1.40), a pacemaker of the glycolytic pathway, catalyzes irreversibly the transphosphorylation from P-enolpyruvate to ADP, generating pyruvate and ATP (1, 2). There are four different isozymes (L, R, M 1 , and M 2 ) in mammalian tissues, which differ in their regulatory properties. These isozymes are allosteric in nature with the exception of the M 1 form, present in skeletal muscle and brain (3-7). PKM 2 is a ubiquitous prototype enzyme present in all tissues during the embryonic stage and is gradually replaced by other isozymic forms in specific tissues during development. The M 2 , L, and R isozymes show homotropic cooperative activation with P-enolpyruvate and heterotropic cooperative activation with Fru-1,6-P 2 (8 -10). The M 1 isozyme is regulated by neither P-enolpyruvate nor Fru-1,6-P 2 because of its intrinsic active conformation in the R-state (5, 6). Under unfavorable conditions such as hypoxia and lack of glucose supply, the anaerobic tissues and tumor cells rely heavily on PKM 2 for ATP production (7). Therefore, stringent control of PK activity is of great importance not only for cell metabolism but also for tumorigenic proliferation.The M 1 and M 2 isozymes are produced from a single gene locus by mutually exclusive alternative splicing (11)(12)(13)(14). In the human M 1 and M 2 isozymes, the exon that is exchanged because of alternative splicing encodes 56 amino acids, in which a total of 22 amino acids differ within a length of 45 residues. The residues located in this region form the major intersubunit contact domain (8). The distinguishable kinetic pro...

Dominant Negative Mutations Affect Oligomerization of Human Pyruvate Kinase M2 Isozyme and Promote Cellular Growth and Polyploidy

“…4 It was therefore pertinent to understand the mechanism of the down-regulation of PKM 2 activity and its functional implication to infer the role the isozyme could play in a cell. We have proposed earlier that structural modulation in PKM 2 mutant proteins may facilitate tumor survival, incidentally one of the key features of BS (20). However, it is not clear if a genetically inherent tendency for stabilization of the PKM 2 dimer and other less active oligomeric forms of PKM 2 , as observed in this study, provides a unique way for activity regulation with a possible clue for genetic predisposition to cancer development in BS patients.…”

“…Gel Permeation, Glycerol Gradient, and Glutaraldehyde Cross-linking Assay-To study the effect of co-expression of wild type and mutant PKM 2 on the oligomeric state of the enzyme, 5 mg of co-expressed protein (His-tagged wild type and His-tagged mutant PKM 2 ) was loaded in a Superdex 300 Superfine (Amersham Biosciences) gel permeation column, and the rest of the procedure adopted was the same as described earlier (20). However, to study the possibility of heterotetramerization, a GST-fused monomer of PK-WT (86 kDa) was co-expressed with His-tagged mutant protein (60 kDa) in E. coli to allow for the generation of heterotetramers of various sizes (ranging from 266 -318 kDa) depending upon the stoichiometry of cross-monomer interaction with a possibility of independent wild type and mutant homotetramers (344 and 240 kDa, respectively).…”

“…Protein Purification, Activity Assay, Enzyme Kinetics, and Bacterial Growth Curve-Protein purification from E. coli, estimation, and activity assays were performed as described earlier (20). P-enolpyruvate saturation kinetics was performed by taking an equal amount of co-expressed wild type-wild type and each of the wild type-mutant PKM 2 proteins at various concentrations (0 -5 mM) of P-enolpyruvate.…”

“…P-enolpyruvate saturation kinetics was performed by taking an equal amount of co-expressed wild type-wild type and each of the wild type-mutant PKM 2 proteins at various concentrations (0 -5 mM) of P-enolpyruvate. The initial velocity data were fit to the Michaelis-Menten equation for calculating values of K m and V max as described earlier (20). Growth curve of BL21DE3 E. coli, co-expressing GST-fused PK-WT and Histagged mutant proteins (from pGEX-4T1 and pET28a(ϩ), respectively), was made by inducing the protein expression at 0 h and measuring A 600 of the culture at every 2-h interval.…”

“…One of the mutants, H391Y, had shown a significant structural rigidity, thermostability, stability over a range of pH, and a loss of typical allosteric behavior. We hypothesized how a multifunctional protein modulates its structure and regulates its function to adapt to stressful conditions like cancer to favor tumor promotion (20). Because the mutations observed were dominant negative in nature and were observed in vivo under heterozygous conditions, it was pertinent to understand the nature of interaction between wild type and mutant proteins under bi-allelic expression conditions.…”