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

Mir Hussain Nawaz,Juliana C Ferreira,Serdal Kirmizialtin,Lyudmila Nedyalkova,Wael M Rabeh,Haizhong Zhu,César Carrasco-López

doi:10.1042/bsr20171666

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.

Highlights

A high rate of glucose metabolism is essential to support the rapid growth of different types of tumors that utilize aerobic glycolysis rather than oxidative phosphorylation, a phenomenon known as the Warburg effect [1,2,3]
HK1 and hexokinase 2 (HK2) of the four human hexokinase isozymes bind to the outer mitochondrial membrane (OMM) via their mitochondrial-binding peptide (MBP) at the N-terminus [24]
When bound to the mitochondria, the MBP points into the mitochondria away from the HK2 enzyme

Summary

Introduction

A high rate of glucose metabolism is essential to support the rapid growth of different types of tumors that utilize aerobic glycolysis rather than oxidative phosphorylation, a phenomenon known as the Warburg effect [1,2,3]. The elevated rates of glucose metabolism, tumor progression, and the high patient mortality has been predominantly associated with the overexpression of HK2 in different cancers [6]. This phenotype has been utilized clinically for the diagnosis of cancer by Positron Emission Tomography (PET) scan [7]. Computational modeling showed the linker helix-α13 to remotely modulate glucose binding by controlling the grove width of the N-domain active site. These findings can explain the inactivation of the N-half of HK1 with similar active site residues to HK2 but different conformation of its linker helix. These results indicate the importance of the N-domain in controlling the stability of the whole enzyme in the addition to its role in binding HK2 to the mitochondria

Experimental procedures

Results

Discussion