Background For a long time cancer cells are known for increased uptake of glucose and its metabolization through glycolysis. and immunoprecipitation (IP) experiment respectively. Interacting domains of GAPDH and its associated proteins were assessed by molecular docking analysis. Mechanism of MG mediated GAPDH inactivation in SDF-5 cancer cells was evaluated by measuring enzyme activity Circular dichroism (CD) spectroscopy IP and mass spectrometry analyses. Result Here we report that GAPDH is associated with glucose-6-phosphate isomerase (GPI) and pyruvate kinase M2 (PKM2) in Ehrlich ascites carcinoma (EAC) cells and also in 3-methylcholanthrene (3MC) induced mouse tumor tissue. Molecular docking analyses suggest C-terminal domain preference for the interaction between GAPDH and GPI. However both C and N termini of PKM2 might be interacting with the C terminal domain of GAPDH. Expression of both PKM2 and GPI is increased in 3MC induced tumor compared with the normal tissue. In presence of 1 1?mM MG association of GAPDH with PKM2 or GPI is not perturbed but the enzymatic activity of GAPDH is reduced to 26.8?±?5?% in 3MC induced tumor and 57.8?±?2.3?% in EAC cells. Treatment of MG to purified GAPDH complex leads to glycation at R399 residue of PKM2 only and changes the secondary structure of the protein complex. Conclusion PKM2 may regulate the Rebaudioside C enzymatic activity of GAPDH. Increased enzymatic activity of GAPDH in tumor cells may be attributed to its association with PKM2 Rebaudioside C and GPI. Association of GAPDH with PKM2 Rebaudioside C and GPI could be a signature for cancer cells. Glycation at R399 of PKM2 and changes in the secondary structure of GAPDH complex could be one of the mechanisms by which GAPDH activity is inhibited in tumor cells by MG. Electronic supplementary material The online version of this article (doi:10.1186/s12885-016-2172-x) contains supplementary material which is available to authorized users. Fig.?2a). We quantified the expression of each of the enzyme and Fig.?2b shows that expression of GPI is higher by 2.2?±?0.45 fold whereas as that GAPDH is lower by 1.8?±?0.22 in 3MC induced tumor compared with normal tissue. On the other hand PKM2 was not detectable in normal tissue. Christofk et al.  have recently shown that PKM2 but not PKM1 (another alternative spliced isoform of PKM) is advantageous for tumor cell growth and critical for tumorigenesis. We checked the expression of PKM1 in 3MC induced tumor tissue. Additional file 1: Figure S2 shows that PKM1 is detectable only in normal tissue but not in the 3MC induced tumor tissue suggesting that tumor tissue expresses only PKM2. Fig. 2 Expression profile of three enzymes in mouse normal and 3MC induced tumor tissues. a Lysates were subjected to immunoblot analysis using Rebaudioside C anti-PKM2 (panel 1) ?GPI (panel 2) ?GAPDH (panel 3) and β-tubulin (panel 4) antibodies. … Association of GAPDH with PKM2 and GPI in tumor cell was validated by immunoprecipitation assay. We immunoprecipitated GAPDH in normal and 3MC induced tumor tissue lysates using antibody against GAPDH and the precipitate was further probed with antibodies against PKM2 GPI and GAPDH. In Fig.?3a (and Additional file 1: Figure S3A-B) panels 1 and 2 show that both PKM2 and GPI are detectable in the immunoprecipitate of GAPDH antibody but not of mouse IgG in 3MC induced tumor tissue (molecular docking analysis. 3D structure of human GAPDH (PDB code: 1U8F chain O) was docked onto PKM2 (PDB code: 1ZJH chain A) and GPI (PDB code: 1JLH chain A) independently without providing any prior information to the docking programs. Top docking solutions from each programs ClusPro [28 29 PatchDock  and SwarmDock  were screened and pooled together for interface analysis. Figure?4 and Additional file 1: Figure S4 plot the overall and average frequencies of N or C terminal domain/residue involvement of GAPDH PKM2 and GPI proteins within the GAPDH-PKM2 (Fig.?4 and Additional file 1: Figure S4A-C) and GAPDH-GPI (Fig.?4 and Additional file 1: Figure S4D-F) docking complexes respectively. Frequencies of C terminal domain of GAPDH are significantly higher in GAPDH-PKM2 (Fig.?4b) and GAPDH-GPI (Fig.?4e) docking complexes advocating the role of C terminal part of GAPDH in.