# Glucose Isomerase: Functions, Structures, and Applications
## 3 Structure
### Metal-Binding State at the GI Active Site  
With regard to metal binding, the active site of GI can exist in three different states: (i) two-metal-binding state, (ii) one-metal-binding state, and (iii) metal free-state (Figure 5) [23; 33; 40]. Although GI crystal structures exhibit different metal-binding states at their active site, the overall GI fold is almost similar, indicating that metal binding does not influence the overall protein folding [40].  
In the two-metal- binding state of GI, the M1 and M2 sites at active site are occupied by divalent metal ions such as Mg\({}^{2+}\), Mn\({}^{2+}\), and Co\({}^{2+}\), and the differences in enzyme activity occur depending on the metal occupying the active site [41; 42]. In the crystal structure of the GI from _S. rubiginosus_, determined via cryo-crystallography, the metal ion at the M1 site is coordinated by Glu181, Glu217, Asp245, and Asp287, as well as the glycerol or ethylene glycol molecule, which is used as a cryoprotectant [33; 40]. Meanwhile, in the room-temperature structure of _S. rubiginosus_ GI, the metal ion at the M1 site is coordinated by the conserved metal-binding residues and water molecules [23; 37], and the metal ion at the M2 site is coordinated by the conserved Glu217, His220, Asp255 (binate interaction), Asp257, and water molecules. The distance between the metal-binding residues and the metal is shorter at the M1 site than at the M2 site, indicating that the M1 site has tighter metal-protein interactions than the M2 site.  
In the one-metal-binding mode of GI, the metal ion exists only at the M1 site involved in substrate recognition [33], indicating the higher affinity of the M1 site for metal binding than the M2 site. This result is consistent with the tighter interaction previously observed between the metal-binding residues and the metal at the M1 site, than the interaction at the M2 site [37]. In the one-metal-binding mode, the sugar substrate or inhibitor can interact with the metal ion at the M1 site [33]. However, isomerization activity through a hydride shift mechanism cannot occur due to the absence of a metal ion at the M2 site, leading to an inactive state of GI.  
In the metal-free state of GI, no metal is found at both the M1 and M2 sites [40]. In this state, the substrate cannot bind to the active site because neither the metal binding to the M2 site involved in the isomerization activity nor the metal of the M1 site to which the substrate binds exist. In the metal-free state, conformation changes in conserved metal-binding residues occur within 1 A despite the absence of metal ions at the two metal sites [40]. This is attributed to stabilization of the position of the metal-binding residues by the neighboring residues around the active sites (Glu181 *-Asn215, Glu217 *-Asn247, His220 *-Pro182, Asp245 *-Asn215, Asp245 *-His285, Asp255 *-Lys183, Asp257 *-Asn247, Asp257 *-Lys289, and Asp287 *-Trp16; metal-binding residues are indicated by an asterisk),  
Figure 5: Three different metal-binding states in the active site of the GI from _Streptomyces rubiginosus_ (SruGI). (**a**) Two-metal-binding mode (PDB code: 6 IRK), (**b**) one-metal-binding mode (PDB code: 5Y4I), and (**c**) metal-free state of SruGI (PDB code: 7 CJP).  
which helps them maintain their positions without large conformational changes [40]. Accordingly, the metal-free state exhibits a minimum open configuration for the metal to perceive and bind to the sites [40]. Based on the existing biochemical knowledge, this study proposed that, when combining with GI in a metal-free state, the metal first binds to the M1 site and then is filled in the M2 site.