Monophosphate Reductase Research Articles

Effects of Curcumin Treatment on Cell Energy Status, Levels of Mitochondrial Enzymes, and Gene Expression of Glucose-related Mechanism in Pancreatic Cancer Cell Lines

Background and Purpose:. Curcumin is an active component of turmeric, has antitumor, immunomodulatory, anti-inflammatory effects. It was aimed to investigate the effects of the administration of curcumin on the energy metabolism, the abnormal redox defense mechanism profile, the malignant transformation indicator of Panc-1 and BxPC-3 pancreatic cancer cells. 
 Methods: BxPC-3 and Panc-1 cells were incubated, were replaced with containing various concentrations of curcumin (10-125 μM) for 24 h. Cell lysate Adenosine triphosphate (ATP), Adenosine diphosphate (ADP), Adenosine monophosphate (AMP), Manganese superoxidase (MnSOD), and cytochrome p450 reductase (CPR) concentrations were analyzed with HPLC and ELISA methods. Genes expression of Lactate dehydrogenase (LDH), mitochondrially encoded ATP synthase membrane subunit 6 (MTATP6), Glucose transporter 1 (GLUT1), and cytochrome p450 were analyzed. 
 Results and Conclusion: IC50 values for 24 hours were found as 47,26 μM in BxPC-3 and 45,84 μM in Panc-1 cells. Treatment with curcumin inhibits oxidative stress by increasing MnSOD enzyme levels. ATP levels did not change in BxPC-3 cells, but it showed an increase in Panc-1 supplemented with curcumin. The effects of curcumin on GLUT-1 are significantly important at a dose of curcumin of 45 μM concentration and affect glucose consumption in both cells. Curcumin showed anti-proliferative, and antioxidant effects.

The mycobacterial guaB1 gene encodes a guanosine 5'-monophosphate reductase with a cystathionine-β-synthase domain.

Mycobacteria express enzymes from both the de novo and purine-salvage pathways. However, the regulation of these processes and the roles of individual metabolic enzymes have not been sufficiently detailed. Both Mycobacterium tuberculosis (Mtb) and Mycobacterium smegmatis (Msm) possess three guaB genes, but information is only available on guaB2, which encodes an essential inosine 5'-monophosphate dehydrogenase (IMPDH) involved in de novo purine biosynthesis. This study shows that guaB1, annotated in databases as a putative IMPDH, encodes a guanosine 5'-monophosphate reductase (GMPR), which recycles guanosine monophosphate to inosine monophosphate within the purine-salvage pathway and contains a cystathionine-β-synthase domain (CBS), which is essential for enzyme activity. GMPR activity is allosterically regulated by the ATP/GTP ratio in a pH-dependent manner. Bioinformatic analysis has indicated the presence of GMPRs containing CBS domains across the entire Actinobacteria phylum.

Phosphorylation of guanosine monophosphate reductase triggers a GTP-dependent switch from pro- to anti-oncogenic function of EPHA4

Signal transduction pathways post-translationally regulating nucleotide metabolism remain largely unknown. Guanosine monophosphate reductase (GMPR) is a nucleotide metabolism enzyme that decreases GTP pools by converting GMP to IMP. We observed that phosphorylation of GMPR at Tyr267 is critical for its activity and found that this phosphorylation by ephrin receptor tyrosine kinase EPHA4 decreases GTP pools in cell protrusions and levels of GTP-bound RAC1. EPHs possess oncogenic and tumor-suppressor activities, although the mechanisms underlying switches between these two modes are poorly understood. We demonstrated that GMPR plays a key role in EPHA4-mediated RAC1 suppression. This supersedes GMPR-independent activation of RAC1 by EPHA4, resulting in a negative overall effect on melanoma cell invasion and tumorigenicity. Accordingly, EPHA4 levels increase during melanoma progression and inversely correlate with GMPR levels in individual melanoma tumors. Therefore, phosphorylation of GMPR at Tyr267 is a metabolic signal transduction switch controlling GTP biosynthesis and transformed phenotypes.

Structural and Dynamical Impact of Water Molecules at Substrate- or Product-Binding Sites in Human GMPR Enzyme: A Study by Molecular Dynamics Simulations.

Human guanosine monophosphate reductase (hGMPR) enzyme maintains the intracellular balance between adenine and guanine nucleotide pools, and it is an excellent target for the design of isoform-specific antileukemic agents. In the present study, we have investigated solvation properties of substrate GMP or product inosine-5'-monophosphate (IMP)-binding pocket of hGMPR by employing molecular dynamics simulations on conformations A (substrate GMP), B [substrate GMP with cofactor nicotinamide adenine dinucleotide phosphate (NDP)], C (product IMP with cofactor NDP), and D (product IMP). Nineteen water sites are identified precisely; they are responsible for the catalytic activity of this site, control structural and dynamical integrity, and electronic consequences of GMP or IMP in the binding site of hGMPR. The water sites of category-1 (W1, W4, W5, W6, W13, and W15) in normal protein and category-2 (W2, W3, W7, W8, W10, W17, and W18) in cancerous protein are unique and stabilize the guanosine or inosine group of GMP or IMP for participation in the enzymatic reaction, whereas the remaining water centers either stabilize pentose sugar ribose or the phosphate group of GMP or IMP. Furthermore, water sites of category-4 (W11, W14, and W16) appear to be conserved in all conformations during the entire simulation. The GMP-binding site in cancerous protein 2C6Q is significantly expanded, and its dynamics are very different from normal protein 2BLE. Furthermore, unique interactions of GMP(N1)···W2···Asp129/Asn158, IMP(N1)···W3···Glu289, and IMP(O6)···W10···Ser270 might be used in a water mimic drug design for hGMPR-II. In this context, water finding probability, relative interaction energy (J) associated with water site W, entropy, and topologies of these three water sites are thermodynamically acceptable for the water displacement method by the modified ligand. Hence, their positions in the catalytic pocket may also facilitate future drug discovery for chronic myelogenous leukemia by the design of appropriately oriented chemical groups that may displace these water molecules to mimic their structural, electronic, and thermodynamic properties.

Enzyme-Substrate-Cofactor Dynamical Networks Revealed by High-Resolution Field Cycling Relaxometry.

The remarkable power and specificity of enzyme catalysis rely on the dynamic alignment of the enzyme, substrates, and cofactors, yet the role of dynamics has usually been approached from the perspective of the protein. We have been using an underappreciated NMR technique, subtesla high-resolution field cycling 31P NMR relaxometry, to investigate the dynamics of the enzyme-bound substrates and cofactor on guanosine-5'-monophosphate reductase (GMPR). GMPR forms two dead end, yet catalytically competent, complexes that mimic distinct steps in the catalytic cycle: E·IMP·NADP+ undergoes a partial hydride transfer reaction, while E·GMP·NADP+ undergoes a partial deamination reaction. A different cofactor conformation is required for each partial reaction. Here we report the effects of mutations designed to perturb cofactor conformation and ammonia binding with the goal of identifying the structural features that contribute to the distinct dynamic signatures of the hydride transfer and deamination complexes. These experiments suggest that Asp129 is a central cog in a dynamic network required for both hydride transfer and deamination. In contrast, Lys77 modulates the conformation and mobility of substrates and cofactors in a reaction-specific manner. Thr105 and Tyr318 are part of a deamination-specific dynamic network that includes the 2'-OH of GMP. These residues have comparatively little effect on the dynamic properties of the hydride transfer complex. These results further illustrate the potential of high-resolution field cycling NMR relaxometry for the investigation of ligand dynamics. In addition, exchange experiments indicate that NH3/NH4+ has a high affinity for the deamination complex but a low affinity for the hydride transfer complex, suggesting that the movement of ammonia may gate the cofactor conformational change. Collectively, these experiments reinforce the view that the enzyme, substrates, and cofactor are linked in intricate, reaction-specific, dynamic networks and demonstrate that distal portions of the substrates and cofactors are critical features in these networks.

Structural Studies of GMP Reductase from Escherichia coli

Guanosine Monophosphate Reductase (GMPR) is an NADPH‐dependent enzyme that catalyzes the reductive deamination of guanosine (G) monophosphate nucleotides into their corresponding inosine (I) monophosphate derivatives. GMPR plays an important role in purine salvage, re‐utilization of free intracellular bases, and contributes overall to the intracellular balance of G and adenine (A) nucleotides. As with many other enzymes in the purine salvage or biosynthesis pathways, GMPR is widespread in nature across eukaryotic and prokaryotic taxa. Given the interest in purine metabolism as a potential target for drug design, GMPR has been characterized both structurally and kinetically in different studies using GMPR derived from several species. We report progress towards solving the crystal structure of GMPR from Escherichia coli in order to extend the insight from existing studies of the E. coli GMPR kinetic and thermodynamic characteristics. Additionally, lessons learned from integrating related research activities into an undergraduate laboratory course will be presented in order to promote adoption of similar course‐based research practices at other primarily‐undergraduate institutions.Support or Funding InformationIthaca College Department of ChemistryThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Dynamic Characteristics of Guanosine-5'-monophosphate Reductase Complexes Revealed by High-Resolution 31P Field-Cycling NMR Relaxometry.

The ability of enzymes to modulate the dynamics of bound substrates and cofactors is a critical feature of catalysis, but the role of dynamics has largely been approached from the perspective of the protein. Here, we use an underappreciated NMR technique, subtesla high-resolution field-cycling 31P NMR relaxometry, to interrogate the dynamics of enzyme bound substrates and cofactors in guanosine-5'-monophosphate reductase (GMPR). These experiments reveal distinct binding modes and dynamic profiles associated with the 31P nuclei in the Michaelis complexes for the deamination and hydride transfer steps of the catalytic cycle. Importantly, the substrate is constrained and the cofactor is more dynamic in the deamination complex E·GMP·NADP+, whereas the substrate is more dynamic and the cofactor is constrained in the hydride transfer complex E·IMP·NADP+. The presence of D2O perturbed the relaxation of the 31P nuclei in E·IMP·NADP+ but not in E·GMP·NADP+, providing further evidence of distinct binding modes with different dynamic properties. dIMP and dGMP are poor substrates, and the dynamics of the cofactor complexes of dGMP/dIMP are disregulated relative to GMP/IMP. The substrate 2'-OH interacts with Asp219, and mutation of Asp219 to Ala decreases the value of Vmax by a factor of 30. Counterintuitively, loss of Asp219 makes both substrates and cofactors less dynamic. These observations suggest that the interactions between the substrate 2'-OH and Asp219 coordinate the dynamic properties of the Michaelis complexes, and these dynamics are important for progression through the catalytic cycle.

Guanosine monophosphate reductase 1 is a potential therapeutic target for Alzheimer\u2019s disease

Alzheimer’s disease (AD) is a severe neurodegenerative disorder for which identification of differentially expressed genes is one way to find new therapeutic targets. Here, we conducted analysis to identify age-independent, AD-specific genes. We found that the MET, WIF1, and NPTX2 genes are downregulated in AD. WIF1 and MET are implicated in Wnt and MET signaling and regulate GSK3β activity and are thus linked with AD. Importantly, we found that the GMPR gene exhibited a gradual increase in AD progression. A logistic model based on GMPR has good ability to classify AD cases. GMPR’s product GMPR1 is in the AMPK and adenosine receptor pathways and is thus associated with Tau phosphorylation in AD. This allows GMPR1 to be a therapeutic target. Therefore, we screened five possible inhibitors to GMPR1 by docking GMPR1 with 1,174 approved drugs. Among them, lumacaftor is ideal. We then tested the effects of lumacaftor on AD model mice. After 20 days of oral administration, we observed that β-Amyloid accumulation was slowed down, and phosphorylation of Tau was almost eliminated in the treated mice. We highlight the elevated expression level of GMPR in AD and propose a therapeutic strategy of inhibiting GMPR1 with lumacaftor.

CA19-9 response combined with CT-imaging accurately predicts resectability of locally advanced pancreatic carcinoma following neoadjuvant chemotherapy

Trypanosoma congolense is one of the most prevalent pathogens which causes trypanosomosis in African animals, resulting in a significant economic loss. In its life cycle, T. congolense is incapable of synthesizing purine nucleotides via a de novo pathway, and thus relies on a salvage pathway to survive. In this study, we identified a gene from T. congolense, TcIL3000_5_1940, as a guanosine 5′-monophosphate reductase (GMPR), an enzyme that modulates the concentration of intracellular guanosine in the pathogen. The recombinant protein was expressed in Escherichia coli, and the gene product was enzymatically confirmed as a unique GMPR, designated as rTcGMPR. This enzyme was constitutively expressed in glycosomes at all of the parasite's developmental stages similar to other purine nucleotide metabolic enzymes. Mycophenolic acid (MPA) was found to inhibit rTcGMPR activity. Hence, it is a potential lead compound for the design of trypanocidal agents, specifically GMPR inhibitor.

Identification and characterization of guanosine 5′-monophosphate reductase of Trypanosoma congolense as a drug target

Trypanosoma congolense is one of the most prevalent pathogens which causes trypanosomosis in African animals, resulting in a significant economic loss. In its life cycle, T. congolense is incapable of synthesizing purine nucleotides via a de novo pathway, and thus relies on a salvage pathway to survive. In this study, we identified a gene from T. congolense, TcIL3000_5_1940, as a guanosine 5′-monophosphate reductase (GMPR), an enzyme that modulates the concentration of intracellular guanosine in the pathogen. The recombinant protein was expressed in Escherichia coli, and the gene product was enzymatically confirmed as a unique GMPR, designated as rTcGMPR. This enzyme was constitutively expressed in glycosomes at all of the parasite's developmental stages similar to other purine nucleotide metabolic enzymes. Mycophenolic acid (MPA) was found to inhibit rTcGMPR activity. Hence, it is a potential lead compound for the design of trypanocidal agents, specifically GMPR inhibitor.

Substrate and Cofactor Dynamics on Guanosine Monophosphate Reductase Probed by High Resolution Field Cycling 31P NMR Relaxometry

Guanosine-5'-monophosphate reductase (GMPR) catalyzes the reduction of GMP to IMP and ammonia with concomitant oxidation of NADPH. Here we investigated the structure and dynamics of enzyme-bound substrates and cofactors by measuring 31P relaxation rates over a large magnetic field range using high resolution field cycling NMR relaxometry. Surprisingly, these experiments reveal differences in the low field relaxation profiles for the monophosphate of GMP compared with IMP in their respective NADP+ complexes. These complexes undergo partial reactions that mimic different steps in the overall catalytic cycle. The relaxation profiles indicate that the substrate monophosphates have distinct interactions in E·IMP·NADP+ and E·GMP·NADP+ complexes. These findings were not anticipated by x-ray crystal structures, which show identical interactions for the monophosphates of GMP and IMP in several inert complexes. In addition, the motion of the cofactor is enhanced in the E·GMP·NADP+ complex. Last, the motions of the substrate and cofactor are coordinately regulated; the cofactor has faster local motions than GMP in the deamination complex but is more constrained than IMP in that complex, leading to hydride transfer. These results show that field cycling can be used to investigate the dynamics of protein-bound ligands and provide new insights into how portions of the substrate remote from the site of chemical transformation promote catalysis.

Microphthalmia-associated transcription factor suppresses invasion by reducing intracellular GTP pools.

Melanoma progression is associated with increased invasion and, often, decreased levels of microphthalmia-associated transcription factor (MITF). Accordingly, downregulation of MITF induces invasion in melanoma cells, however little is known about the underlying mechanisms. Here, we report for the first time that depletion of MITF results in elevation of intracellular GTP levels and increased amounts of active (GTP-bound) RAC1, RHO-A and RHO-C. Concomitantly, MITF-depleted cells display larger number of invadopodia and increased invasion. We further demonstrate that the gene for guanosine monophosphate reductase (GMPR) is a direct MITF target, and that the partial repression of GMPR accounts mostly for the above phenotypes in MITF-depleted cells. Reciprocally, transactivation of GMPR is required for MITF-dependent suppression of melanoma cell invasion, tumorigenicity, and lung colonization. Moreover, loss of GMPR accompanies downregulation of MITF in vemurafenib-resistant BRAFV600E-melanoma cells and underlies the increased invasion in these cells. Our data uncover novel mechanisms linking MITF-dependent inhibition of invasion to suppression of guanylate metabolism.

The cystathionine-β-synthase domains on the guanosine 5''-monophosphate reductase and inosine 5'-monophosphate dehydrogenase enzymes from Leishmania regulate enzymatic activity in response to guanylate and adenylate nucleotide levels.

The Leishmania guanosine 5'-monophosphate reductase (GMPR) and inosine 5'-monophosphate dehydrogenase (IMPDH) are purine metabolic enzymes that function maintaining the cellular adenylate and guanylate nucleotide. Interestingly, both enzymes contain a cystathionine-β-synthase domain (CBS). To investigate this metabolic regulation, the Leishmania GMPR was cloned and shown to be sufficient to complement the guaC (GMPR), but not the guaB (IMPDH), mutation in Escherichia coli. Kinetic studies confirmed that the Leishmania GMPR catalyzed a strict NADPH-dependent reductive deamination of GMP to produce IMP. Addition of GTP or high levels of GMP induced a marked increase in activity without altering the Km values for the substrates. In contrast, the binding of ATP decreased the GMPR activity and increased the GMP Km value 10-fold. These kinetic changes were correlated with changes in the GMPR quaternary structure, induced by the binding of GMP, GTP, or ATP to the GMPR CBS domain. The capacity of these CBS domains to mediate the catalytic activity of the IMPDH and GMPR provides a regulatory mechanism for balancing the intracellular adenylate and guanylate pools.

Novel Characteristics of Trypanosoma brucei Guanosine 5'-monophosphate Reductase Distinct from Host Animals.

The metabolic pathway of purine nucleotides in parasitic protozoa is a potent drug target for treatment of parasitemia. Guanosine 5’-monophosphate reductase (GMPR), which catalyzes the deamination of guanosine 5’-monophosphate (GMP) to inosine 5’-monophosphate (IMP), plays an important role in the interconversion of purine nucleotides to maintain the intracellular balance of their concentration. However, only a few studies on protozoan GMPR have been reported at present. Herein, we identified the GMPR in Trypanosoma brucei, a causative protozoan parasite of African trypanosomiasis, and found that the GMPR proteins were consistently localized to glycosomes in T. brucei bloodstream forms. We characterized its recombinant protein to investigate the enzymatic differences between GMPRs of T. brucei and its host animals. T. brucei GMPR was distinct in having an insertion of a tandem repeat of the cystathionine β-synthase (CBS) domain, which was absent in mammalian and bacterial GMPRs. The recombinant protein of T. brucei GMPR catalyzed the conversion of GMP to IMP in the presence of NADPH, and showed apparent affinities for both GMP and NADPH different from those of its mammalian counterparts. Interestingly, the addition of monovalent cations such as K+ and NH4+ to the enzymatic reaction increased the GMPR activity of T. brucei, whereas none of the mammalian GMPR’s was affected by these cations. The monophosphate form of the purine nucleoside analog ribavirin inhibited T. brucei GMPR activity, though mammalian GMPRs showed no or only a little inhibition by it. These results suggest that the mechanism of the GMPR reaction in T. brucei is distinct from that in the host organisms. Finally, we demonstrated the inhibitory effect of ribavirin on the proliferation of trypanosomes in a dose-dependent manner, suggesting the availability of ribavirin to develop a new therapeutic agent against African trypanosomiasis.

GTP Metabolism Regulates Cancer Cell Invasion

Invasion is a prerequisite for metastasis which is the most detrimental feature of virtually all types of cancers. Tumor cell invasion has been strongly associated with the activity of small GTPases, in particular those of the RHO-GTPase family. The question of whether tumor cells possess intrinsic ability to regulate activity of the above GTPases and subsequently invasion by manipulating intracellular GTP pools has never been addressed. We demonstrate that a purine nucleotide metabolism enzyme guanosine monophosphate reductase (GMPR) suppresses the ability of highly invasive melanoma cells to invade in vitro and grow as tumor xenografts in vivo. Mechanistically, GMPR partially depleted intracellular GTP pools and reduced the amounts of several GTP-bound (active) RHO-GTPases. shRNA-mediated inhibition of GMPR or addition of exogenous guanosine increased activity of the same RHO-GTPases and up-regulated invasion in melanoma cells and cells from different types of cancers. Ectopic expression of constitutively active RAC1G12V, insensitive to GTP depletion, substantially reverted GMPR-dependent phenotypes. The functional role of intracellular GTP levels in tumor cell invasion was confirmed by inhibiting activity of other enzymes involved in the de novo guanylate biosynthesis. Our data identified a previously unrecognized ability of cancer cells to increase the activity of RHO-GTPases via up-regulation of GTP pools, and established a novel and fundamental connection between tumor cell invasion and metabolism of guanylates

Identification of The Binding Site for Ammonia in GMP Reductase

The reaction of guanosine monophosphate reductase (GMPR) includes two steps: (1) a deamination step that releases ammonia from GMP and forms the intermediate E‐XMP*; (2) a hydride transfer step that converts E‐XMP* to IMP utilizing NADPH as a cofactor. The hydride transfer step is the rate limiting step, yet we failed to observe a burst of ammonia release. This observation suggests that ammonia remains bound to the enzyme during the hydride transfer step. We identified a possible ammonia binding site by inspection of crystal structure of human GMPR type 2. Three candidate amino acids were selected and probed by site directed mutagenesis. The substitution of all 3 residues decreased intermediate production about 4 fold but decreased the reaction with ammonia by at least 100 fold. Therefore, these substitutions behave as expected for mutations at the ammonia binding site.

A Purine Nucleotide Biosynthesis Enzyme Guanosine Monophosphate Reductase Is a Suppressor of Melanoma Invasion

Melanoma is one of the most aggressive types of human cancers, and the mechanisms underlying melanoma invasive phenotype are not completely understood. Here, we report that expression of guanosine monophosphate reductase (GMPR), an enzyme involved in de novo biosynthesis of purine nucleotides, was downregulated in the invasive stages of human melanoma. Loss- and gain-of-function experiments revealed that GMPR downregulates the amounts of several GTP-bound (active) Rho-GTPases and suppresses the ability of melanoma cells to form invadopodia, degrade extracellular matrix, invade invitro, and grow as tumor xenografts invivo. Mechanistically, we demonstrated that GMPR partially depletes intracellular GTP pools. Pharmacological inhibition of de novo GTP biosynthesis suppressed whereas addition of exogenous guanosine increased invasion of melanoma cells as well as cells from other cancer types. Our data identify GMPR as a melanoma invasion suppressor and establish a link between guanosine metabolism and Rho-GTPase-dependent melanoma cell invasion.

Cofactor mobility determines reaction outcome in the IMPDH and GMPR (β-α)8 barrel enzymes

IMP dehydrogenase (IMPDH) and GMP reductase (GMPR) belong to the same structural family, share a common set of catalytic residues and bind the same ligands. The structural and mechanistic features that determine reaction outcome in the IMPDH/GMPR family have not been identified. Here, we show that the GMPR reaction utilizes the same intermediate E-XMP* as IMPDH, but this intermediate reacts with ammonia instead of water. A single crystal structure of human GMPR type 2 with IMP and NADPH fortuitously captures three different states, each of which mimic a distinct step in the catalytic cycle of GMPR. The cofactor is found in two conformations, an "in" conformation poised for hydride transfer, and an "out" conformation where the cofactor is 6 Å from IMP. Mutagenesis, substrate/cofactor analog experiments demonstrate that the “out” conformation is required for the deamination of GMP. Remarkably, the cofactor is part of the catalytic machinery activating ammonia.

Guanosine monophosphate reductase regulates uncoupling protein 1 activity

Non-shivering thermogenesis is mediated by brown adipose tissue (BAT) specific uncoupling protein 1 (UCP1). Activity of UCP1 is regulated by free fatty acids and by the di- and triphosphate forms of purine nucleotides. GMP reductase (GMPR) interconverts guanosine into adenosine nucleotides and is upregulated in response to cold. We engineered human embryonic kidney (HEK) cells to stably express UCP1 (HEK-UCP1). In HEK and HEK-UCP1 cells we transiently overexpressed GMPR and measured the concentration of all purine nucleotides. In HEK cells GMPR expression did not lead to altered concentrations of nucleotides, while in HEK-UCP1 cells GMPR decreased the ratio of triphosphate to mono- and diphosphate purine nucleotides, indicating a lowered capacity for oxidative phosphorylation. Upon GMPR expression in HEK-UCP1 both the basal proton leak (in the presence of substrate plus oligomycin) and the palmitate-inducible, UCP1-specific proton leak was greater than in the absence of either UCP1, GMPR or both. This implies an augmentation of UCP1 activity by GMPR. Surprisingly, we were able to several-fold enrich GMPR by isolation of mitochondrial protein from BAT. Possibly, GMPR is associated with mitochondria and is implicated in the maintenance of a microdomain of altered purine concentrations to modify UCP1 activity, as reflected in our respiration and nucleotide concentration measurements.

Recombinant Escherichia coli GMP reductase: kinetic, catalytic and chemical mechanisms, and thermodynamics of enzyme–ligand binary complex formation

Guanosine monophosphate (GMP) reductase catalyzes the reductive deamination of GMP to inosine monophosphate (IMP). GMP reductase plays an important role in the conversion of nucleoside and nucleotide derivatives of guanine to adenine nucleotides. In addition, as a member of the purine salvage pathway, it also participates in the reutilization of free intracellular bases. Here we present cloning, expression and purification of Escherichia coli guaC-encoded GMP reductase to determine its kinetic mechanism, as well as chemical and thermodynamic features of this reaction. Initial velocity studies and isothermal titration calorimetry demonstrated that GMP reductase follows an ordered bi-bi kinetic mechanism, in which GMP binds first to the enzyme followed by NADPH binding, and NADP(+) dissociates first followed by IMP release. The isothermal titration calorimetry also showed that GMP and IMP binding are thermodynamically favorable processes. The pH-rate profiles showed groups with apparent pK values of 6.6 and 9.6 involved in catalysis, and pK values of 7.1 and 8.6 important to GMP binding, and a pK value of 6.2 important for NADPH binding. Primary deuterium kinetic isotope effects demonstrated that hydride transfer contributes to the rate-limiting step, whereas solvent kinetic isotope effects arise from a single protonic site that plays a modest role in catalysis. Multiple isotope effects suggest that protonation and hydride transfer steps take place in the same transition state, lending support to a concerted mechanism. Pre-steady-state kinetic data suggest that product release does not contribute to the rate-limiting step of the reaction catalyzed by E. coli GMP reductase.