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 AMPbound 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.Glucose metabolism is central to the biochemistry of all living systems with the enzymatic phosphorylation of glucose playing a key role in cellular energy metabolism by ensuring that this energy-rich substrate is available to the cell. A relatively recently discovered ADP-dependent glucokinase (ADPGK) 3 (EC 2.7.1.147) catalyzes the phosphorylation of D-glucose to glucose-6-phosphate using MgADP as phosphoryl donor in contrast to the more typical ATP-utilizing hexokinases and glucokinases. ADPGK was first identified in Archaea, being involved in a modified Embden-Meyerhof glycolytic pathway (1), in which Archaea can also use an ADP-dependent phosphofructokinase (ADPPFK; EC 2.7.1.146). Bioinformatic analysis led to the identification of ADPGK in metazoa and the subsequent cloning and initial characterization of mammalian ADPGKs (2, 3). Mammalian ADPGKs show modest sequence similarity to archaeal orthologues (ϳ20% amino acid identity). ADPGK is highly expressed in a wide variety of both normal and tumor mammalian tissues (3) and has been found to be localized to the endoplasmic reticulum in T cells (4) consistent with the sequence-based annotation of an N-terminal signal peptide. Furthermore, ADPGK has been identified as a cholesterol binding protein in a proteomics screen (5). Despite the role of archaeal ADPGK in glycolysis, overexpression of ADPGK in H460 and HC116 human tumor cells showed no cell proliferative or glycolytic effects (3). ADPGK knock-out in t...

Hypothalamic arcuate nucleus glucokinase regulates insulin secretion and glucose homeostasis

“…Glucokinase (GK) is a member of the hexokinase family with important roles in glucose sensing and disposal. There are two isoforms of GK, a hepatic form, expressed exclusively in the liver, and a neuroendocrine form expressed in pancreatic β cells and the central nervous system (CNS) .…”

Structural basis for ADP-dependent glucokinase inhibition by 8-bromo–substituted adenosine nucleotide

“…In higher eukaryotes, this step is catalyzed by several tissue type-specific isoforms of ATP-utilizing enzymes known as hexokinases (HKs; 4 isoforms I to IV in human) (3). An alternative glycolytic pathway involving ADP-dependent glucokinase (ADPGK) (EC 2.7.1.147) was first described in hyperthermophilic Archaea (4), but ADPGK homologs were subsequently discovered and functionally characterized in mouse (mADPGK) (3) and human (hADPGK) (5,6). ADPGKs are structurally unrelated to HKs and constitute distant homologs of family B of sugar kinases within the ribokinase superfamily (7).…”

Crystal structure of ADP‐dependent glucokinase from Methanocaldococcus jannaschii in complex with 5‐iodotubercidin reveals phosphoryl transfer mechanism

“…8 Recently, an experimental structure of a N-terminally truncated variant of murine ADPGK (mADPGK) in the apo form and in complex with AMP (PDB codes 5CCF, 5CK7) has been published. 9 Despite little sequence homology (20% identity), the overall fold of mADPGK is similar to that of the archaeal ADPGKs and ADP-PFKs. In fact, the similarity is so high, that it allowed us to solve the mADPGK structure by molecular replacement using the archaeal protein as a search model.…”

“…Despite the lack of sequence similarity between ADPGK and ATP‐dependent kinases, the structure of ADPGK is very similar to those of ribokinase (PDB:) and adenine kinase (PDB:) . Recently, an experimental structure of a N‐terminally truncated variant of murine ADPGK (mADPGK) in the apo form and in complex with AMP (PDB codes , ) has been published . Despite little sequence homology (20% identity), the overall fold of mADPGK is similar to that of the archaeal ADPGKs and ADP‐PFKs.…”