The catalytic inactivation of the N-half of human hexokinase 2 and structural and biochemical characterization of its mitochondrial conformation

Abstract

The high proliferation rate of tumor cells demands high energy and metabolites that are sustained by a high glycolytic flux known as the ‘Warburg effect’. The activation and further metabolism of glucose is initiated by hexokinase, a focal point of metabolic regulation. The human hexokinase 2 (HK2) is overexpressed in all aggressive tumors and predominantly found on the outer mitochondrial membrane, where interactions through its N-terminus initiates and maintains tumorigenesis. Here, we report the structure of HK2 in complex with glucose and glucose-6-phosphate (G6P). Structural and biochemical characterization of the mitochondrial conformation reveals higher conformational stability and slow protein unfolding rate (ku) compared with the cytosolic conformation. Despite the active site similarity of all human hexokinases, the N-domain of HK2 is catalytically active but not in hexokinase 1 and 3. Helix-α13 that protrudes out of the N-domain to link it to the C-domain of HK2 is found to be important in maintaining the catalytic activity of the N-half. In addition, the N-domain of HK2 regulates the stability of the whole enzyme in contrast with the C-domain. Glucose binding enhanced the stability of the wild-type (WT) enzyme and the single mutant D657A of the C-domain, but it did not increase the stability of the D209A mutant of the N-domain. The interaction of HK2 with the mitochondria through its N-half is proposed to facilitate higher stability on the mitochondria. The identification of structural and biochemical differences between HK2 and other human hexokinase isozymes could potentially be used in the development of new anticancer therapies.

The Warburg Effect 97 Years after Its Discovery

“…Therefore, the MPC complex is a regulator of glycolysis in tumor cells as, under hypoxic conditions, lactate secretion from cancer cells increases, while MPC1 and MPC2 levels decrease [ 60 ]. The human HK2, overexpressed in all aggressive tumors, is predominantly located in the outer mitochondrial membrane, where the interaction through its N-terminus increases its stability and maintains tumorigenesis [ 26 , 61 ]. The predominant role of HK2 in tumor cells is also confirmed by the observation that the tumor subgroups expressing both HK1 and HK2 are sensitive to inhibition of HK2 alone [ 62 ].…”

Biochemical and Biophysical characterization of the main protease, 3-chymotrypsin-like protease (3CLpro), from the novel coronavirus disease 19 (COVID-19)

“…It has been shown previously that proteins with dominated hydrophilic structural interactions exhibit an exothermic positive thermographic peak compared with hydrophobic interactions that exhibit an endothermic negative thermographic peak. 19 The reduced ΔHcal value at pH 11.0 is an indication of a structural fold with dominated hydrophobic interactions compared to the hydrophilic character of the protein at the other pH values tested here.…”

“…A.) as described previously 19 . Similar to DSC, the Tm of 3CLpro was measured at different ionic strength in the presence or absence of 250 mM NaCl or 250 mM MgCl2 in 50mM phosphate buffer at pH 7.5.…”

Linker residues regulate the activity and stability of hexokinase 2, a promising anticancer target

“…Thermodynamic measurements were performed on the FL variants of the single mutants D447A, S449A, and K451A in the absence or presence of glucose. Previously, we reported an increase in the thermodynamic stability of HK2 variants upon glucose binding (12). From DSC analysis, all thermal melts of HK2 were irreversible with a second heating scan included for each run as outlined in the methods section.…”

“…1D). The catalytic residues D209 and D657 are required to deprotonate the hydroxyl on C6 of glucose in preparation for its nucleophilic attack on the g-phosphate of ATP (7,(12)(13)(14).…”