Research Article: The phosphoglycerate kinase 1 variants found in carcinoma cells display different catalytic activity and conformational stability compared to the native enzyme

Date Published: July 11, 2018

Publisher: Public Library of Science

Author(s): Annarita Fiorillo, Maria Petrosino, Andrea Ilari, Alessandra Pasquo, Alessandra Cipollone, Maristella Maggi, Roberta Chiaraluce, Valerio Consalvi, Jose M. Sanchez-Ruiz.


Cancer cells are able to survive in difficult conditions, reprogramming their metabolism according to their requirements. Under hypoxic conditions they shift from oxidative phosphorylation to aerobic glycolysis, a behavior known as Warburg effect. In the last years, glycolytic enzymes have been identified as potential targets for alternative anticancer therapies. Recently, phosphoglycerate kinase 1 (PGK1), an ubiquitous enzyme expressed in all somatic cells that catalyzes the seventh step of glycolysis which consists of the reversible phosphotransfer reaction from 1,3-bisphosphoglycerate to ADP, has been discovered to be overexpressed in many cancer types. Moreover, several somatic variants of PGK1 have been identified in tumors. In this study we analyzed the effect of the single nucleotide variants found in cancer tissues on the PGK1 structure and function. Our results clearly show that the variants display a decreased catalytic efficiency and/or thermodynamic stability and an altered local tertiary structure, as shown by the solved X-ray structures. The changes in the catalytic properties and in the stability of the PGK1 variants, mainly due to the local changes evidenced by the X-ray structures, suggest also changes in the functional role of PGK to support the biosynthetic need of the growing and proliferating tumour cells.

Partial Text

Carcinomas are the most common type of cancer. According to the American Cancer Society, in 2016 the overall estimate is of 1685210 new cases of cancer worldwide and among them 61000 cases of female breast carcinoma in situ are expected [1]. Cancer cells are able to adapt to survive in difficult conditions, like for example in O2 deficiency, through a reprogramming of their metabolic machinery according to their requirements [2]. Among these, the hallmark of the metabolic reprogramming is the shift from oxidative phosphorylation to aerobic glycolysis that allows tumor cells to survive under hypoxic conditions [3], an adaptative behaviour described nearly 100 years ago and known as the Warburg effect. In the last years, glycolytic enzymes, and their role in cancer metabolism, have been the object of several studies and they have been identified as potential targets for alternative anticancer therapies [4, 5].

In this study we selected seven PGK1 variants (R38M, R65W, G166D, M189I, A199V, V216F and F241S) mined from the COSMIC database ( [24] and associated to human carcinoma. Most of these PGK1 variants, M189I, A199V, V216F and F241S (Fig 1), are reported to be found in breast carcinoma; the other variants, R38M, R65W and G166D are reported to be present in lung, liver and endometrium carcinoma. The location of the selected mutants mapped onto the PGK1 structure is shown in Fig 1. Residues R38, R65 and G166 are located in the enzyme 3-PG binding site (Fig 1) at the N-terminal domain; in particular, the residues R38 and G166 are part of two helices, whereas the residue R65 is located in a loop (Fig 1). R38 and R65 are involved in 3-PG binding; in particular, R65 NH1 and NH2 groups are involved in an electrostatic interaction with the carboxyl group of 3-PG, whereas R65 Nε and NH2 groups acquire contacts with the phosphate group of 3-PG [43]. G166 is located in the 3-PG binding pocket interacting with the substrate C2 through its main carboxylic group. M189 and A199 are located at the edges of the helix that represents the flexible hinge region that allow the two domains of the enzyme to approach each other during the catalytic cycle (Fig 1). Residue V216 is located in a turn, in proximity of the nucleotide-binding A214 residue; F241 is part of a helix, and they both belong to the MgADP binding pocket, located at the protein C-terminal domain (Fig 1). Among the mutations studied, only R65W involves a surface exposed residue, all others variants, R38M, G166D, M189I, A199V, V216F and F241S are more buried. We generated recombinant protein for each of the identified mutants using site directed mutagenesis and available bacterial expression systems. Introduction of these mutations resulted in soluble recombinant proteins and allowed us to study the consequences of the mutations on PGK1 thermal and thermodynamic stability and the kinetic activity.

Phosphoglycerate kinase 1 (PGK1) is an ubiquitous enzyme expressed in all somatic cells that plays a central role in glycolysis where it provides energy in form of ATP through the reversible phosphotransfer reaction from 1,3-bisphosphoglycerate (1,3-BPG) to MgADP in order to produce 3-phosphoglycerate (3-PG) and MgATP in the presence of free magnesium. PGK1 is a central enzyme in the cancer cells metabolism since they utilize preferentially glycolysis to produce ATP. Alteration of PGK1 levels has been reported in different cancer types and this enzyme is considered as a negative prognostic marker [23]. In the general metabolic reprogramming of cancer cells, glycolytic enzymes play a pivotal role since many intermediates of the glycolytic pathway may also be utilized for the increased biosynthetic demand of proliferating tumor cells [49]. Indeed, one of the consequence of the metabolic change in cancer cells is the request of reducing equivalent for biosynthesis that can be provided through the deviation of glycolysis intermediate in the shunt of pentose phosphate pathway [50].




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