Biochemical and structural insights into how amino acids regulate pyruvate kinase muscle isoform 2

Abstract

Pyruvate kinase muscle isoform 2 (PKM2) is a key glycolytic enzyme involved in ATP generation and critical for cancer metabolism. PKM2 is expressed in many human cancers and is regulated by complex mechanisms that promote tumor growth and proliferation. Therefore, it is considered an attractive therapeutic target for modulating tumor metabolism. Various stimuli allosterically regulate PKM2 by cycling it between highly active and less active states. Several small molecules activate PKM2 by binding to its intersubunit interface. Serine and cysteine serve as an activator and inhibitor of PKM2, respectively, by binding to its amino acid (AA)-binding pocket, which therefore represents a potential druggable site. Despite binding similarly to PKM2, how cysteine and serine differentially regulate this enzyme remains elusive. Using kinetic analyses, fluorescence binding, X-ray crystallography, and gel filtration experiments with asparagine, aspartate, and valine as PKM2 ligands, we examined whether the differences in the side-chain polarity of these AAs trigger distinct allosteric responses in PKM2. We found that Asn (polar) and Asp (charged) activate PKM2 and that Val (hydrophobic) inhibits it. The results also indicate that both Asn and Asp can restore the activity of Val-inhibited PKM2. AA-bound crystal structures of PKM2 displayed distinctive interactions within the binding pocket, causing unique allosteric effects in the enzyme. These structure-function analyses of AA-mediated PKM2 regulation shed light on the chemical requirements in the development of mechanism-based small-molecule modulators targeting the AA-binding pocket of PKM2 and provide broader insights into the regulatory mechanisms of complex allosteric enzymes.

Purine nucleotide depletion prompts cell migration by stimulating the serine synthesis pathway

“…4l ). Given that the K d of PKM2 for ADP is 200–300 µM 16 , its activity would be sensitive to this degree of ADP depletion. Indeed, we found that loss of Pkm2 (Δ Pkm2 ) in MEFs exhibit an increased shunt from glycolysis into serine synthesis relative to wild-type MEFs, and MTX treatment failed to increase serine flux in these cells despite a robust depletion of purine nucleotides Fig.…”

[Retracted] Mangifera indica Extracts as Novel PKM2 Inhibitors for Treatment of Triple Negative Breast Cancer

“…The X-ray crystallography structure of the human pyruvate kinase M2 (PKM2) was obtained from the https://www.rcsb.org/structure6V74 [ 15 ]. Proteins were imported to a Molegro Virtual Docker [ 16 ] and prepared for docking.…”

miR-1307 promotes hepatocarcinogenesis by CALR-OSTC-endoplasmic reticulum protein folding pathway

“…Carcinogenic Ras mutations have three isotypes: HRAS, KRAS, and NRAS, which are the background of many tumorigenesis ( Kumazaki et al., 2020 ). PKM2 is highly expressed in a variety of human tumor cells, which provides favorable conditions for the survival, growth, and diffusion of tumor cells ( Nandi and Dey, 2020 ). PLK1 is overexpressed in a variety of human cancers, which can directly participate in the cell-cycle regulation of tumor cells ( Goroshchuk et al., 2020 ).…”