5shows NCI with values for some critical points within the quantum region. substitution of type 2 (SN2) with a mix of SN1-type molecular mechanism. Based on our structural analysis, a magnesium-assisted SN2-type mechanism would be involved in the reverse reaction. These results provide a framework for understanding the molecular mechanism and substrate discrimination in both directions by APRTs. This knowledge can play an instrumental role in the design of inhibitors, such as antiparasitic brokers, or adenine-based substrates. or through salvage pathways by specific enzymes. Two such enzymes in the salvage pathway are adenine phosphoribosyltransferase (APRT, EC 2.4.2.7)4 and hypoxanthine-guanine phosphoribosyltransferase (HGPRT, EC 2.4.2.8). These are enzymes with reversible activities having an ordered sequential bi-bi reaction mechanism (1, 2). In the forward reaction, APRT synthesizes AMP from adenine (ADE) (Fig. 1), whereas HGPRT generates GMP or IMP from guanine (Gua) or hypoxanthine (Hx), respectively. Both reactions use the co-substrate -d-5-phosphoribosyl-1-pyrophosphate (PRPP) and at least one divalent magnesium ion. In the reverse pathway, PPi and the corresponding ribonucleoside monophosphate are substrates of the reaction (8). Similarly to other phosphoribosyltransferase enzymes, both APRT and HGPRT structures are made of a Rossmann fold. They also include a PRPP-binding motif, a flexible loop, and a hood region Lck Inhibitor (Fig. S1). The last seems to provide purine specificity, either ADE or Hx and Gua, in APRT or HGPRT, respectively (3, 4). Furthermore, the flexible loop is very dynamic (5), and we recently showed that a conserved tyrosine within the flexible loop of human APRT facilitates the forward reaction and is essential for cell growth (6). Open in a separate window Physique 1. Reversible enzymatic reactions by hAPRT. and Note S1), remains in the active site and interacts with the conserved Ala-131CTGGTCPRPPCbinding motif (Figs. 2and S1). A network of water molecules replaced the ribose and pyrophosphate moieties and interacts with the conserved Arg-67, Asp-127, Asp-128, Ala-131 and Gly-133 residues (Fig. Lck Inhibitor 2(?)47.4, 47.6, 47.749.1, 49.8, 71.847.6, 47.6, 47.947.4, 47.7, 47.8????????, , ()77.1, 69.4, 61.790.1, 93.2, 102.376.4, 69.2, 61.376.7, 69.3, 61.5????Resolution (?)1.701.901.521.55????Quantity of molecules in asymmetric models2422????Measured reflections78,393112,869175,07898,424????Redundancy? ?is the intensity of the hkl reflection; ?Calculated using Molprobity. Open in a separate window Physique 2. Structure of phosphate-bound hAPRT. C omit electron density map for any phosphate ion contoured at 3. Without substrate bound in the active site, the conserved Arg-67 adopted two conformations. or in the phosphate- or PRPP-bound structures, respectively. All the figures were generated with PyMOL. The hydrogen bonds are represented with throughout. Five of these water molecules (called a, b, c, d, e) were located in close vicinity of the six oxygen atoms coordinating the magnesium ion in the PRPP-Mg2+-hAPRT structures (PDB IDs: 6FCH, 6FCI, and 6FD4) (Fig. 2? omit density map contoured at 3 for Hx is usually shown in Fig. 3? omit map densities for PRPP showed full occupancy for this substrate (Fig. 3and C omit electron density map for Hx (increased by 2.5 C as compared with a 9.4 C increase with AMP) (Table 1). Combined, the two substrates (PPi and AMP) potentiated the stabilization of the enzyme (of 64.3 C with = 14.7 C). Stabilization of the enzyme was also observed with IMP and GMP, although to a lesser extent. The increased only 2C3 C with the addition of IMP or GMP to the enzyme as compared with substrate-free hAPRT. Moreover, addition of PPi with IMP or GMP to the enzyme did not increase the values. Therefore, the complexation of IMP and GMP to hAPRT seems less favorable than with AMP, and PPi may not contribute to the binding. To probe how IMP or GMP interact with hAPRT, we decided the crystal structures of the two complexes, IMP-hAPRT and GMP-hAPRT (Table 2). We diffused the IMP and GMP molecules into substrate-free hAPRT crystals and elucidated the structures to a better than 1.6 ? resolution. The structures were then compared with the natural AMP-hAPRT complex (PDB ID: 6FCL). The electron densities in the active sites were readily attributable to IMP and GMP (Fig. 4, and and Fig. S3). Even though the interactions with the phosphate and the sugar moieties were conserved,.Similarly to other phosphoribosyltransferase enzymes, both APRT and HGPRT structures Lck Inhibitor are made of a Rossmann fold. in the forward reaction, whereas it is base-specific in the reverse reaction. Furthermore, Lck Inhibitor a quantum mechanics/molecular mechanics (QM/MM) analysis suggests that the forward reaction is mainly a nucleophilic substitution of type 2 (SN2) with a mix of SN1-type molecular mechanism. Based on our structural analysis, a magnesium-assisted SN2-type mechanism would be involved in the reverse reaction. These results provide a framework for understanding the molecular mechanism and substrate discrimination in both directions by APRTs. This knowledge can play an instrumental role in the design of inhibitors, such as antiparasitic brokers, or adenine-based substrates. or through salvage pathways by specific enzymes. Two such enzymes in the salvage pathway are adenine phosphoribosyltransferase (APRT, EC 2.4.2.7)4 and hypoxanthine-guanine phosphoribosyltransferase (HGPRT, EC 2.4.2.8). These are enzymes with reversible activities having an ordered sequential bi-bi reaction mechanism (1, 2). In the forward reaction, APRT synthesizes AMP from adenine (ADE) (Fig. 1), whereas HGPRT generates GMP or IMP from guanine (Gua) or hypoxanthine (Hx), respectively. Both reactions use the co-substrate -d-5-phosphoribosyl-1-pyrophosphate (PRPP) and at least one divalent magnesium ion. In the reverse pathway, PPi and the corresponding ribonucleoside monophosphate are substrates of the reaction (8). Similarly to other phosphoribosyltransferase enzymes, both APRT and HGPRT structures are made of a Rossmann fold. They also include a PRPP-binding motif, a flexible loop, and a hood region (Fig. S1). The last seems to provide purine specificity, either ADE or Hx and Gua, in APRT or HGPRT, respectively (3, 4). Furthermore, the flexible loop is very dynamic (5), and we recently showed that a conserved tyrosine within the flexible loop of human APRT facilitates the forward reaction and is essential for cell growth (6). Open in a separate window Physique 1. Reversible enzymatic reactions by hAPRT. and Note S1), remains in the active site and interacts with the conserved Ala-131CTGGTCPRPPCbinding motif (Figs. 2and S1). A network of water molecules replaced the ribose and pyrophosphate moieties and interacts with the conserved Arg-67, Asp-127, Asp-128, FGD4 Ala-131 and Gly-133 residues (Fig. 2(?)47.4, 47.6, 47.749.1, 49.8, 71.847.6, 47.6, 47.947.4, 47.7, 47.8????????, , ()77.1, 69.4, 61.790.1, 93.2, 102.376.4, 69.2, 61.376.7, 69.3, 61.5????Resolution (?)1.701.901.521.55????Quantity of molecules in asymmetric models2422????Measured reflections78,393112,869175,07898,424????Redundancy? ?is the intensity of the hkl reflection; ?Calculated using Molprobity. Open in a separate window Physique 2. Structure of phosphate-bound hAPRT. C omit electron density map for any phosphate ion contoured at 3. Without substrate bound in the active site, the conserved Arg-67 adopted two conformations. or in the phosphate- or PRPP-bound structures, respectively. All the figures were generated with PyMOL. The hydrogen bonds are represented with throughout. Five of these water molecules (called a, b, c, d, e) were located in close vicinity of the six oxygen atoms coordinating the magnesium ion in the PRPP-Mg2+-hAPRT structures (PDB IDs: 6FCH, 6FCI, and 6FD4) (Fig. 2? omit density map contoured at 3 for Hx is usually shown in Fig. 3? omit map densities for PRPP showed full occupancy for this substrate (Fig. 3and C omit electron density map for Hx (increased by 2.5 C as compared with a 9.4 C increase with AMP) (Table 1). Combined, the two substrates (PPi and AMP) potentiated the stabilization of the enzyme (of 64.3 C with = 14.7 C). Stabilization of the enzyme was also observed with IMP and GMP, although to a lesser extent. The increased only 2C3 C with the addition of IMP or GMP to the enzyme as compared with substrate-free hAPRT. Moreover, addition of PPi with IMP or GMP to the enzyme did not increase the values. Therefore, the complexation of IMP and GMP to hAPRT seems less favorable than with AMP, and PPi may not contribute to the binding. To probe how IMP or GMP interact with hAPRT, we decided the crystal structures of the two complexes, IMP-hAPRT and GMP-hAPRT (Table 2). We diffused the IMP and GMP molecules into substrate-free hAPRT crystals and elucidated the structures to a better than 1.6 ? resolution. The structures were then compared with the natural AMP-hAPRT complex (PDB ID: 6FCL). The electron densities in the active sites were readily attributable to IMP and GMP (Fig. 4, and and Fig. S3). Even though the interactions with the phosphate and the sugar moieties were conserved, the base was differently situated. Specifically, with AMP, the N1 atom faces the main chain NH group of Arg-27 and presents its lone electron pair to form a hydrogen bond of 3.0 ? (Fig. S3). In the IMP complex, the N1 atom is at a hydrogen bond distance of 2.8 ? from your carboxyl group of Arg-27 (Fig. 4and C omit electron density maps for IMP (and.