The Structural and Functional Characterization of Mammalian ADP-dependent Glucokinase

Abstract

The enzyme-catalyzed phosphorylation of glucose to glucose-6-phosphate is a reaction central to the metabolism of all life. ADP-dependent glucokinase (ADPGK) catalyzes glucose-6-phosphate production, utilizing ADP as a phosphoryl donor in contrast to the more well characterized ATP-requiring hexokinases. ADPGK is found in Archaea and metazoa; in Archaea, ADPGK participates in a glycolytic role, but a function in most eukaryotic cell types remains unknown. We have determined structures of the eukaryotic ADPGK revealing a ribokinase-like tertiary fold similar to archaeal orthologues but with significant differences in some secondary structural elements. Both the unliganded and the AMP-bound ADPGK structures are in the "open" conformation. The structures reveal the presence of a disulfide bond between conserved cysteines that is positioned at the nucleotide-binding loop of eukaryotic ADPGK. The AMP-bound ADPGK structure defines the nucleotide-binding site with one of the disulfide bond cysteines coordinating the AMP with its main chain atoms, a nucleotide-binding motif that appears unique to eukaryotic ADPGKs. Key amino acids at the active site are structurally conserved between mammalian and archaeal ADPGK, and site-directed mutagenesis has confirmed residues essential for enzymatic activity. ADPGK is substrate inhibited by high glucose concentration and shows high specificity for glucose, with no activity for other sugars, as determined by NMR spectroscopy, including 2-deoxyglucose, the glucose analogue used for tumor detection by positron emission tomography.

Keywords: ADP; ADP-dependent glucokinase; AMP; NMR spectroscopy; enzyme structure; glucokinase; glucose metabolism; glucose-6-phosphate; ribokinase; x-ray crystallography.

FIGURE 1.

The structure of mouse ADPGK. Shown is a ribbon representation of the mADPGKΔ51 structure color-ramped blue to red from the N to C termini. The large (orange) and the small (yellow) nucleotide-binding loops are labeled. The disulfide bond is displayed as sticks with sulfurs in yellow (Cys-414–Cys-469), and other key amino acid side chains are displayed in stick representation colored by atom type.

FIGURE 2.

ADPGK structure-based sequence alignment. PromalS3D sequence alignment based on the structure of mouse ADPGK and the structures of archaeal ADPGKs. Helices (α, η), sheets (β), and turns (TT) in the structure of mADPGK are labeled. The mADPGK disulfide bond Cys are marked with green triangles. hADPGK residues targeted for site-directed mutagenesis are marked with red stars. mm, M. musculus; hs, Homo sapiens; ph, P. horikoshii; pf, P. furiosus; tl, T. litoralis.

FIGURE 3.

Structural superposition of mouse and archaeal ADPGK structures. The superposition of mADPGKΔ51 and T. litoralis ADPGK (PDB code 4B8R) in ribbon representation. Regions distinctly different from the archaeal ADPGK on the small (α2) and large (α1 and β17/18) are colored (gray/blue for mADPGK and yellow/red for T. litoralis ADPGK).

FIGURE 4.

Mouse ADPGK nucleotide binding. A, the orientation and color coding of the structural representation are similar to Fig. 1. AMP and selected side chains are shown in stick representation color-coded by atom type. B, electron density around the AMP bound to ADPGK. The electron density is displayed as a blue mesh (contoured at 1.0 σ) and clipped to the AMP molecule. C, electron density defining the ADPGK disulfide bridge. The electron density is displayed as a blue mesh (contoured at 1.2 σ). D, backbone representation of the apo- (blue) and AMP-bound (red) mADPGK structures. The AMP and the disulfide bond are displayed in green and yellow stick representation, respectively.

FIGURE 5.

Sequence alignment of the C-terminal region of ADPGKs from Eukaryotes and Archaea. hs, H. sapiens; mm, M. musculus; pt, Pan troglodytes; bt, Bos taurus; ec, Equus caballus; oc, Oryctolagus cuniculus; gg, Gallus gallus; dr, Danio rerio; xt, Xenopus tropicalis; ce, Caenorhabditis elegans; sp, Stronglycentrotus purpuratus; si, Solenopsis invicta; bm, Bombyx mori; dm, Drosophila melanogaster; tt, Tetrahymena thermophila; tl, T. litoralis; pf, P. furiosus; ph, P. horikoshii. Cysteine residues homologous to those involved in the formation of the disulfide bond observed in M. musculus ADPGK are highlighted with yellow shading. The disulfide bond cysteines are indicated with a green triangle. The disulfide bond cysteines are highly conserved in Eukarya but absent in Archaea.

FIGURE 6.

Mouse ADPGK glucose binding site. The mouse ADPGK glucose binding site inferred from homology to archaeal ADPGK by structural superposition of mADPGK (gray), and glucose/AMP bound T. litoralis ADPGK (PDB code 4B8S, yellow). The d-glucose (green) from 4B8S is displayed. All labels refer to mADPGK amino acids.

FIGURE 7.

³¹P NMR spectra for screening substrate specificity of human ADPGK. A control reaction with MgADP and d-glucose is shown at the bottom, where the signals for the products AMP and d-glucose-6-phosphate are indicated. No product was detectable for all other spectra and the signal intensity for the α- and β-anomers of ADP remained unchanged. G-6-P, glucose-6-phosphate.

FIGURE 8.

Human ADPGK kinetic analysis. A, the rate of enzymatic activity of hADPGK as a function of increasing concentration of d-glucose. Units (U) are defined as μmol/min. hADPGK showed inhibition at higher substrate concentrations, and the kinetic parameters were determined accordingly. The K_m and K_i for d-glucose were 0.48 mm and 2.9 mm, respectively. R² = 0.95. Error bars show standard deviation. B, the rate of enzymatic activity of hADPGK as a function of increasing concentration of MgADP. The K_m and K_i values for MgADP were 0.56 mm and 9.1 mm, respectively. R² = 0.96. C, enzyme kinetics of hADPGK, product inhibition by AMP as a function of increasing concentration of MgADP. AMP is a competitive inhibitor for MgADP binding to ADPGK with a K_i of 0.50 mm. The legend on the right shows the concentration of AMP. R² = 0.96. The kinetic parameters were determined by nonlinear regression with Prism 6.0.