The crystal structure of rabbit phosphoglucose isomerase complexed with D‐sorbitol‐6‐phosphate, an analog of the open chain form of D‐glucose‐6‐phosphate

Phosphoglucose isomerase (PGI; E.C. 5.3.1.9) is a cytosolic enzyme that catalyzes the reversible isomerization of G6P and F6P. Its activity is important for glycolysis; gluconeogenesis; the pentose phosphate pathway; and the glycosylation of proteins, lipids, and other molecules. Recently, attention to PGI has grown because PGI is a moonlighting protein with a second function outside of the cell. PGI is the same protein as the extracellular growth factors neuroleukin (Chaput et al. 1988; Faik et al. 1988), autocrine motility factor (Watanabe et al. 1996), and differentiation and maturation mediator (Xu et al. 1996). It stimulates antibody secretion, promotes the survival of embryonic spinal neurons in vitro (Gurney et al. 1986a,b), affects an increase in tumor cell motility, and causes the differentiation of human myelogenous leukemia cells (Chiao et al. 1999) and a human myelocytic cell line (HL-60 cells) to terminal monocytic cells (Leung and Chiao 1985; Abolhassani et al. 1990). Recently, PGI also has been found to be the self-antigen in a mouse model of rheumatoid arthritis (Matsumoto et al. 1999; Schaller et al. 2001). One lab has identified antibodies to PGI in patients suffering from rheumatoid arthritis (Schaller et al. 2001). The enzymatic activity of PGI was first described over 70 years ago (Lohmann 1933). Biochemical characterization of PGI enzyme activity by several labs led to a proposed multistep mechanism involving general acid–base catalysis (Rose and O’Connell 1961; Rose 1975, 1981; Willem et al. 1990). Both G6P and F6P are substrates for PGI activity. Because G6P and F6P exist predominantly in their cyclic forms in solution (Swenson and Barker 1971), it is believed that first the cyclic form of G6P or F6P binds to the active site. PGI catalyzes ring opening of the substrate to yield the open chain form. The reaction then proceeds through the isomerization step using acid–base catalysis with proton transfer. PGI is a dimer with 557 amino acids in each subunit and has two equivalent active sites. X-ray crystal structures have been reported of PGI without bound ligand or with a substrate (F6P) or competitive inhibitors of catalytic activity bound in the active site pocket. X-ray crystal structures of mammalian (rabbit, pig, and human) PGI have been solved with 6PGA (Jeffery et al. 2000), 5PAA (Jeffery et al. 2001; Davies and Muirhead 2002; Davies et al. 2003), 5PAH (Arsenieva et al. 2002), or F6P (Lee et al. 2001), or with no bound ligand (Arsenieva and Jeffery 2002; Davies and Muirhead 2002, 2003). A crystal structure of human PGI with a sulfate ion was also reported (Read et al. 2001). Other crystal structures of PGI included two structures from a bacterium, Bacillus stearothermophilus, with no bound ligand (Sun et al. 1999) or complexed with 5PAA (Chou et al. 2000). Three crystal structures of PGI enzyme from Pyrococcus furiosus were also solved with no bound ligand (Berrisford et al. 2003), with 5PAA, and with 6PGA (Swan et al. 2003). Interestingly, the PGI enzyme from P. furiosus does not share sequence or structural homology with either mammalian or B. stearothermophilus PGI, and its activity is probably the result of convergent evolution. The X-ray crystal structures of PGI have helped to identify active site amino acids, ordered water molecules, and conformational changes involved in the ligand binding, ring opening, and isomerization steps of the multistep catalytic mechanism. Comparison of a crystal structure of rabbit PGI without a bound ligand to structures with ligands provided information about induced fit of the enzyme upon substrate binding. A crystal structure of rabbit PGI complexed with F6P provided information about the roles of Glu216, His388, Lys518, and an ordered water molecule in the ring opening step (Lee et al. 2001). Crystal structures of rabbit PGI complexed with 5PAA and 5PAH were used to identify the role of Glu357 as the active site amino acid residue that transfers a proton between C1 and C2 during the isomerization step (Jeffery et al. 2001) and the role of a water molecule in transferring a proton between the hydroxyl groups on C1 and C2 (Arsenieva et al. 2002). Arg272 was proposed to have a role in helping stabilize the cis-enedio-l(ate) intermediate. Comparison of the PGI/5PAA, PGI/5PAH, and PGI/F6P complexes also indicated that the substrate undergoes a rotation about the C3–C4 bond between the ring opening and isomerization steps to position C1 and C2 near to Glu357. Also, a helix containing amino acid residues 512–520 moves in toward the substrate to help hold it in place for the isomerization step. However, less clear is the state of the active site and substrate between the ring opening and isomerization steps. To complete the details of the catalytic mechanism of PGI, a structure of PGI complexed with an open chain substrate, or an analog, is required. Here, we report an X-ray crystal structure of rabbit PGI complexed with an analog of the open chain form of G6P (S6P), bound in the active site. This structure was solved at 2.0 A resolution.