Study on the Expressions of PHD and HIF in Placentas from Normal Pregnant Women and Patients with Preeclampsia

Objective: To investigate the relationship between oxygen sensitivity of trophoblast and hypoxia in preeclamptic placenta by the study on the expressions of hypoxia-inducible factor prolyl 4-hydroxylase (PHD) and hypoxia-inducible factor (HIF) in placentas from normal pregnant women and patients with pre-eclampsia.Methods: Subjects were chosen from the in-patients or the out-patients from May 2003 to May 2004. They were divided into 5 groups: early pregnancy group (EP), 13 cases; middle pregnancy group (MP), 9 cases; late pregnancy group (LP, or control group), 12 cases; preeclampsia (PE) group, 20 cases; gestational hypertension group (GH), 10 cases. The mRNA expressions of PHD-1 and -2 and -3 in placentas from all the subjects were assessed by in situ hybridization and Real-time PCR. The expressions of HIF-1α and -2α in placentas from different groups were assessed by immunohistochemistry and western blot.Results: PHD-1,-2 and -3 mRNA were mainly expressed in cytoplasm of trophoblast, especially strongly expressed in extravillous trophoblast. During the progress of pregnancy, the expression of PHD-1 increased significantly (R=0.616, P<0.001). The PHD-1mRNA expression in placentas from PE group decreased significantly compared with that from control group, P<0.05. A significant direct correlation between the PHD-1 mRNA expression in placentas from PE group and their placenta weight was found (R=0.457, P<0.05). The HIF-2α, not the HIF-1α expression, from PE group was significantly higher than that from control group, P<0.01; The HIF-2α expression in trophoblast from PE was inversely correlated to the date of the onset of the disease (R=-0.730, P<0.01).Conclusions: PHD-1 played an important role in hypoxic response pathway of trophoblast through modulating the level of HIF-2α. The overly activated hypoxic response pathway of trophoblast in preeclamptic placenta, which is manifested as the result of HIF-2α over-expression, is the key point to hypoxic dysfunction of trophoblast.     

A New Method to Estimate Inhibition Percentage of Endogenous Digitalis in Patients with Pre-eclampsia

Background:Pre-eclampsia is an idiopathic pregnancy disorder characterized by appearance proteinuria and hypertension, with poorly understood etiology. It has been linked to a variety of system abnormalities, including ion transport disorders in neonatal, maternal, and placental cell lines. A new method was described to evaluate the inhibition percentage of endogenous digitalis in plasma of pre-eclampsia patients compared with normal pregnancies, with the estimation of sensitivity and specificity of the proposed test.Methods:This was a case-control study consisting of 130 cases that were divided into three groups, 55 normal pregnancies (positive control), 30 non-pregnant women (negative control), and 45 pre-eclampsia (patients). The new method included the estimation of the percentage inhibition of endogenous digitalis by measuring specific enzyme activity of Na-K ATPase for the patient and positive control. The results were analyzed using the statistical package for social sciences (SPSS®) software version 26.0. A p-value of≤ 0.05 was considered significant. Results:In the pre-eclampsia patient, the specific activity of Na-K ATPase was significantly lower with mean= 0.239 mg/g±0.043 compared to the control group which was 0.397 mg/g±0.021, p< 0.001. While the result of inhibition percentage of endogenous digitalis showed significantly higher in the pre-eclampsia patient (mean= 35.852 mg/g %±2.692%) compared to the control group (mean= 17.964%±1.784), with a p< 0.001.Conclusion:Pre-eclampsia is linked with lower erythrocyte sodium pump activity significantly in pre-eclampsia patients than in normal pregnancies. Also, results show the inhibited percentage of endogenous digitalis elevation in patients with pre-eclampsia compared with normal pregnancy.Key Words: Endogenous Digitalis, Hypertension, Inhibition Percentage, Pre-eclampsia     

Circulating Angiogenic Factors and the Risk of Adverse Outcomes among Haitian Women with Preeclampsia

ObjectiveAngiogenic factors are strongly associated with adverse maternal and fetal outcomes among women with preterm preeclampsia (PE) in developed countries. We evaluated the role of angiogenic factors and their relationship to adverse outcomes among Haitian women with PE.ResultsAmong patients with PE, most (24/35) were admitted at term. Adverse outcome rates in PE were much higher among the early onset group compared to the late onset group (100.0% vs. 54.2%, P=0.007). Plasma angiogenic factors were dramatically altered in both subtypes of PE. Angiogenic factors also correlated with adverse outcomes in both subtypes of PE. The median sFlt1/PlGF ratios for subjects with early onset PE with any adverse outcome vs. NHD <=34 weeks with no adverse outcome were 703.1 (146.6, 1614.9) and 9.6 (3.5, 58.6); P<0.001). Among late onset group the median sFlt1/PlGF ratio for women with any adverse outcome was 130.7 (56.1, 242.6) versus 22.4 (10.2, 58.7; P=0.005) in NHD >34 weeks with no adverse outcome.ConclusionPE-related adverse outcomes are common in women in Haiti and are associated with profound angiogenic imbalance regardless of gestational age at presentation.     

Serum Levels of Soluble Fas and Fas Ligand in Iranian Women with Pre-eclampsia

Background:The precise responsible mechanism of pre-eclampsia remains controversial however, recent data suggest a main role of the abnormal activation of the adaptive immune system and Apoptosis. In this study, we have measured serum levels of Fas/Fasl as two important members of extrinsic apoptotic pathway in patient with pre-eclampsia.Methods:207 participants including 99 pre-eclampsia patients and 108 age and sex-matched normal pregnant women were involved in the case-control study. Plasma sample from each participant was collected and stored at –20 °C until batch processing.Serum levels of Fas and Fas ligand were measured by ELISA for each participant including 99 pre-eclampsia patients and 108 normal pregnant women. Following a test of statistical normality, nonparametric data were analyzed by Mann-Whitney.Results:sFas levels in case group was significantly higher than controls; 584 (397-892) pg/ml in cases opposed to 341 (213-602) pg/ml in controls (p value< 0.01). sFasL in pre-eclampsia women was a little lower than controls; 255 (173-318) pg/ml and in case group compared to 265.5 (184-381.5) pg/ml in controls.Conclusion:We have found the increased levels of sFas in patients with pre-eclampsia in compare with the healthy pregnant women. It seems that abnormality in sFAS is related with pre-eclampsia.Key Words: Pre-eclampsia, Pregnancy, sFAS, sFASL, Apoptosis     

Hypoxia induced‐disruption of lncRNA TUG1/PRC2 interaction impairs human trophoblast invasion through epigenetically activating Nodal/ALK7 signalling

Inadequate trophoblastic invasion is considered as one of hallmarks of preeclampsia (PE), which is characterized by newly onset of hypertension (>140/90 mmHg) and proteinuria (>300 mg in a 24‐h urine) after 20 weeks of gestation. Accumulating evidence has indicated that long noncoding RNAs are aberrantly expressed in PE, whereas detailed mechanisms are unknown. In the present study, we showed that lncRNA Taurine upregulated 1 (TUG1) were downregulated in preeclamptic placenta and in HTR8/SVneo cells under hypoxic conditions, together with reduced enhancer of zeste homolog2 (EZH2) and embryonic ectoderm development (EED) expression, major components of polycomb repressive complex 2 (PRC2), as well as activation of Nodal/ALK7 signalling pathway. Mechanistically, we found that TUG1 bound to PRC2 (EZH2/EED) in HTR8/SVneo cells and weakened TUG1/PRC2 interplay was correlated with upregulation of Nodal expression via decreasing H3K27me3 mark at the promoter region of Nodal gene under hypoxic conditions. And activation of Nodal signalling prohibited trophoblast invasion via reducing MMP2 levels. Overexpression of TUG1 or EZH2 significantly attenuated hypoxia‐induced reduction of trophoblastic invasiveness via negative modulating Nodal/ALK7 signalling and rescuing expression of its downstream target MMP2. These investigations might provide some evidence for novel mechanisms responsible for inadequate trophoblastic invasion and might shed some light on identifying future therapeutic targets for PE.     

Endotoxin induced hyperlactatemia and hypoglycemia is linked to decreased mitochondrial phosphoenolpyruvate carboxykinase

AimsPhosphoenolpyruvate carboxykinase (PEPCK) is the rate limiting enzyme for gluconeogenesis, and plays a key role in recycling lactate for glucose production. It is synthesized as two separate isoforms; cytosolic (PEPCK-C, gene code; PCK1) and mitochondrial (PEPCK-M, gene code; PCK2). Previous studies of gluconeogenesis in endotoxemia have focused solely on PCK1. We investigated the relative roles of the two isoforms in hepatic and renal gluconeogenesis in a rat model of endotoxic shock, and in cultured hepatocytes.Main methodsRats were administered lipopolysaccharide (6 mg/kg; LPS) for 6 h. Cultured cells were incubated with lactate (5 mM) with or without tumor necrosis factor alpha (1 – 10 ng/ml). Rat liver and kidney samples as well as cultured cells were subjected to subcellular fractionation to produce mitochondrial and cytosolic fractions for PEPCK activity assay. PCK1 and PCK2 mRNA levels were measured using quantitative RT-PCR.Key findingsIn rat endotoxemia, hepatic PCK2 mRNA and PEPCK-M enzyme activity decreased by 53% and 38%, compared to sham controls. Hepatic PCK1 mRNA levels increased by 44%, but PEPCK-C enzyme activity remained unchanged. The changes in hepatic PEPCK-M coincided with a marked hypoglycemia and hyperlactatemia as well as elevated plasma interleukin 1 beta (IL1beta). Incubation of cultured hepatocytes with TNF-alpha inhibited lactate-induced increases in glucose production, PCK2 mRNA levels and PEPCK-M enzyme activity but had no effect on PCK1 mRNA levels or PEPCK-C activity.SignificanceThese results indicate that decreases in hepatic PEPCK-M play a key role in the manifestation of hyperlactatemia and hypoglycemia in endotoxemia.     

Molecular dynamics simulation of lactate dehydrogenase adsorption onto pristine and carboxylic-functionalized graphene

ABSTRACT

Lactate dehydrogenase (LDH) is a tetrameric enzyme which is composed of two subunits known as LDHA and LDHB, which are encoded by the LDHA and LDHB genes respectively. LDH catalyses the last step in anaerobic glycolysis through the reversible conversion of pyruvate to lactate via coupled oxidation of NADH cofactor. The LDHA plays an important regulatory role in anaerobic glycolysis, by catalysing the final step of the process. Therefore, it is likely that increases in the expression level of LDHA in cancer cells could facilitate the efficiency of anaerobic glycolysis. Measuring the level of serum LDHA is a key step in the diagnosis of many cancer types. In this study, the adsorption, stability, and dynamics of LDHA on the surface of pristine graphene (PG) and carboxylated graphene (COOH-Graphene) were investigated using its molecular dynamics simulation. Variations in root mean square deviation, root mean square fluctuation, solvent accessible surface area and adsorption energy of the LDHA during the simulation were calculated to analyse the effect of PG and COOH-Graphene on the overall conformation of LDHA. Results showed that the adsorption of LDHA on COOH-Graphene is mostly mediated by electrostatic interactions, whereas on the PG, both Van der Waals and π-π interactions are prominent.     

The Association between Intrauterine Inflammation and Spontaneous Vaginal Delivery at Term: A Cross-Sectional Study

Background

Different factors contribute to the onset of labor at term. In animal models onset of labor is characterized by an inflammatory response. The role of intrauterine inflammation, although implicated in preterm birth, is not yet established in human term labor. We hypothesized that intrauterine inflammation at term is associated with spontaneous onset of labor.

Methods/Results

In two large urban hospitals in the Netherlands, a cross-sectional study of spontaneous onset term vaginal deliveries and elective caesarean sections (CS), without signs of labor, was carried out. Placentas and amniotic fluid samples were collected during labor and/or at delivery. Histological signs of placenta inflammation were determined. Amniotic fluid proinflammatory cytokine concentrations were measured using ELISA. A total of 375 women were included. In term vaginal deliveries, more signs of intrauterine inflammation were found than in elective CS: the prevalence of chorioamnionitis was higher (18 vs 4%, p = 0.02) and amniotic fluid concentration of IL-6 was higher (3.1 vs 0.37 ng/mL, p<0.001). Similar results were obtained for IL-8 (10.93 vs 0.96 ng/mL, p<0.001) and percentage of detectable TNF-α (50 vs 4%, p<0.001).

Conclusions

This large cross-sectional study shows that spontaneous term delivery is characterized by histopathological signs of placenta inflammation and increased amniotic fluid proinflammatory cytokines.     

Global DNA methylation patterns in placenta and its association with maternal hypertension in pre-eclampsia

Maternal nutrition is an important determinant of one-carbon metabolism that lies at the heart of intrauterine epigenetic programming. Exchange of nutrients and other vital molecules between the mother and fetus takes place across the placenta and hence may play direct role in fetal programming. Pre-eclampsia (PE) originates in the placenta and altered maternal nutrition may influence epigenetic patterns in the placenta, thereby affecting birth outcome. In the present study, we investigated the global DNA methylation levels in placentas of pre-eclampsia women (i.e., women delivering at term and those delivering preterm) and studied their associations with maternal blood pressure and birth outcome. Increased homocysteine and global DNA methylation levels were seen in the pre-eclampsia group (term and preterm PE) when compared with the normotensive group (p?相似文献   

Association of Proteinuria Threshold in Pre-Eclampsia with Maternal and Perinatal Outcomes: A Nested Case Control Cohort of High Risk Women

Objectives

To evaluate occurrence of adverse maternal and perinatal outcomes with different thresholds of proteinuria (300-499mg and ≥500mg/24 hours) in pre-eclamptic women, comparing outcomes against women with chronic and gestational hypertension.

Design

Secondary analysis of the Vitamins in Pre-Eclampsia Trial.

Setting

25 UK hospitals in ten geographical areas.

Population

946 women with pre-existing risk factors for pre-eclampsia.

Methods

Women with pre-eclampsia and proteinuria 300-499mg/24h (PE300, referent group, n=60) or proteinuria ≥500 mg/24h (PE500, n=161) were compared with two groups of non-proteinuric women with chronic hypertension (CHT, n=615) or gestational hypertension (GH, n=110).

Main Outcome Measures

Maternal: progression to severe hypertension. Perinatal: small for gestational age (SGA) <5^th centile, gestation at delivery.

Results

Severe hypertension occurred more frequently in PE500 (35%) and PE300 (27%) than CHT (5.9%; P≤0.01) and GH (10%; p≤0.001). Gestation at delivery was earlier in PE500 (33.2w) than PE300 (37.3w; P≤0.001), and later in CHT (38.3w; P≤0.05) and GH (39.1w; P≤0.001). SGA infants were more frequent in PE300 (32%) than in CHT (13.3%; P≤0.001) and GH (16.5%; P≤0.05). Women in PE500 were more likely to have a caesarean section than PE300 (78% vs. 48%; P≤0.001), and to receive magnesium sulphate (17% vs. 1.7%, P≤0.05).

Conclusion

Women with PE300 have complication rates above those of women managed as out-patients (GH and CHT), meriting closer surveillance and confirming 300 mg/d as an appropriate threshold for determining in-patient management. Adverse perinatal outcomes are higher still in women with PE500.     

Phosphoenolpyruvate metabolism in Teladorsagia circumcincta: A critical junction between aerobic and anaerobic metabolism

Nematodes which have adapted to an anaerobic lifestyle in their adult stages oxidise phosphoenolpyruvate (PEP) to oxaloacetate rather than pyruvate as the final product of glycolysis. This adaptation involves selective expression of the enzyme phosphoenolpyruvate carboxykinase (PEPCK), instead of pyruvate kinase (PK). However, such adaptation is not absolute in aerobic nematode species. We have examined the activity and kinetics of PEPCK and PK in larvae (L₃) and adults of Teladorsagia circumcincta, a parasite known to exhibit oxygen uptake. Results revealed that PK and PEPCK activity existed in both L₃s and adults. The enzymes had differing affinity for nucleotide diphosphates: while both can utilise GDP, only PK utilised ADP and only PEPCK utilised IDP. In both life cycle stages, enzymes showed similar affinity for PEP. PK activity was predominant in both stages, although activity of this enzyme was lower in adults. When combined, both the activity levels and the enzyme kinetics showed that pyruvate production is probably favoured in both L₃ and adult stages of T. circumcincta and suggest that metabolism of PEP to oxaloacetate is a minor metabolic pathway in this species.     

Characterization of Key Glycolytic and Oxidative Enzymes in Steinernema carpocapsae

The enzyme activities of isocitrate dehydrogenase (ICDH, NADP-specific), lactate dehydrogenase (LDH), malate dehydrogenase (MDH), phosphoenolpyruvate carboxykinase (PEPCK), phosphofructokinase (PFK), pyruvate kinase (PK), and fructose-l,6-bisphosphatase (FBPase) were studied in the third-stage juveniles of Steinernema carpocapsae. Reaction requirements, pH optima, substrate and cofactor kinetic constants were similar to those reported previously from other parasitic helminths with the exception of LDH, which was unstable and could not be characterized for specific activity and kinetic constants. The respective pH optima were 7.5 for ICDH, 8.8 for MDH, 6.5 for PEPCK, 7.3 for PFK, 7.2 for PK, and 7.5 for FBPase. The specific activities for ICDH, MDH, PEPCK, PFK, PK, and FBPase at pH 7.5 were 4.8, 1,300, 22, 25, 35, and 6.8 (nmoles substrate ∙ min⁻¹ ∙ mg protein⁻¹), respectively. In summary, the infective juveniles of S. carpocapsae display the metabolism typical of a facultative aerobe.     

Metabolism of the obligatory aerobic yeast Rhodotorula gracilis

Summary D-Glucose and D-xylose addition to not-growing Rhodotorula gracilis cells brings about alterations in pyruvate kinase and phosphoenolpyruvate carboxykinase activities characteristic for glycolysis and gluconeogenesis, respectively.Abbreviations used PK Pyruvate kinase (EC 2.7.1.40) - PEPCK Phosphoenolpyruvate carboxykinase (EC 4.1.1.32) - PFK Phosphofructokinase (EC 2.7.1.11)     

Effect of adding plyometric training to physical education sessions on specific biomechanical parameters in primary school girls

Objectives:The study aimed to determine the effect of adding a school-based plyometric training program (PMT) to physical education (PE) sessions on the strength, balance, and flexibility in primary school girls.Methods:Students from grades 3-6 were randomized equally to a plyometric or control group. In the control group, students took their regular PE classes twice a week. In the plyometric group, students performed PMT twice a week during the initial 20 minutes of every PE session. The Lido Linea closed kinetic chain isokinetic dynamometer, Star excursion balance test (SEBT), and sit-and-reach test were used to assess muscle strength, balance, and flexibility, respectively, before and after nine weeks of training.Results:The improvement in extension peak force (p=0.04) and extension total work (p<0.001) was more prevalent in the PMT group than in the control group. SEBT scores had improved significantly (p<0.05) for all directions in the PMT group, except in the anterior direction, which was highly significant (p<0.001). Hamstring and lower back flexibility had improved more in the PMT group than in the control group (p<0.001).Conclusion:Adding PMT to regular PE classes has a positive and notable effect on muscle strength, balance, and flexibility in primary school students.     

Deleted in Breast Cancer 1 (DBC1) Protein Regulates Hepatic Gluconeogenesis

Liver gluconeogenesis is essential to provide energy to glycolytic tissues during fasting periods. However, aberrant up-regulation of this metabolic pathway contributes to the progression of glucose intolerance in individuals with diabetes. Phosphoenolpyruvate carboxykinase (PEPCK) expression plays a critical role in the modulation of gluconeogenesis. Several pathways contribute to the regulation of PEPCK, including the nuclear receptor Rev-erbα and the histone deacetylase SIRT1. Deleted in breast cancer 1 (DBC1) is a nuclear protein that binds to and regulates both Rev-erbα and SIRT1 and, therefore, is a candidate to participate in the regulation of PEPCK. In this work, we provide evidence that DBC1 regulates glucose metabolism and the expression of PEPCK. We show that DBC1 levels decrease early in the fasting state. Also, DBC1 KO mice display higher gluconeogenesis in a normal and a high-fat diet. DBC1 absence leads to an increase in PEPCK mRNA and protein expression. Conversely, overexpression of DBC1 results in a decrease in PEPCK mRNA and protein levels. DBC1 regulates the levels of Rev-erbα, and manipulation of Rev-erbα activity or levels prevents the effect of DBC1 on PEPCK. In addition, Rev-erbα levels decrease in the first hours of fasting. Finally, knockdown of the deacetylase SIRT1 eliminates the effect of DBC1 knockdown on Rev-erbα levels and PEPCK expression, suggesting that the mechanism of PEPCK regulation is, at least in part, dependent on the activity of this enzyme. Our results point to DBC1 as a novel regulator of gluconeogenesis.     

Induction of lactate dehydrogenase isozymes by oxygen deficit in barley root tissue

Lactate dehydrogenase (LDH) activity in attached roots of barley and other cereals increased up to 20-fold during several days of severe hypoxia, reaching a maximum of about 2 micromoles per minute per gram fresh weight. In barley, induction of LDH activity was significant at 2.6% O₂ and greatest at 0.06%, the lowest O₂ concentration tested. Upon return to aerobic conditions, induced LDH activity declined with an apparent half-life of 2 days. The isozyme profile of barley LDH comprised 5 bands, consistent with a tetrameric enzyme with subunits encoded by two different Ldh genes. Changes in staining intensity of the isozymes as a function of O₂ level suggested that one Ldh gene was preferentially expressed in severe hypoxia. When tracer [U-¹⁴C]glucose was supplied to induced roots under hypoxic conditions, lactate acquired label, but much less than either ethanol or alanine. Most of the [¹⁴C] lactate was secreted into the medium, whereas most other labeled anionic products were retained in the root. Neither hypoxic induction of LDH, nor lactate secretion by induced roots, is predicted from the Davies-Roberts hypothesis, which holds that lactate glycolysis ceases soon after the onset of hypoxia due to acidosis brought about by lactate accumulation in the cytoplasm. These results imply a functional significance for LDH beyond that assigned it in this hypothesis.     

Carbohydrate energy metabolism in Fasciola gigantica (trematoda)

Umezurike G. M. and Anya A. O. 1980. Carbohydrate energy metabolism in Fasciola gigantica (Trematoda). International Journal for Parasitology10: 175–180. Adult Fasciola gigantica contained 4.49 ± 0.06 % (mean ± S.D.) wet weight glycogen. Tissue homogenates contained high levels of malate dehydrogenase (MDH), NAD-linked malic enzyme (ME), Phosphoenolpyruvate carboxykinase (PEPCK) and lactate dehydrogenase (LDH). MDH, PEPCK and ME activities appeared to be localized in both cytosolic and mitoehondrial fractions, fumarase activity appeared to be predominantly mitochondrial whereas LDH and pyruvate kinase activities were cytosolic in distribution. Polyacrylamide gel electrophoresis revealed the predominance of LDH-1, LDH-2 and LDH-3 but only traces of LDH-4 and LDH-5 isoenzymes in the crude cytosolic fraction. LDH activity in the crude sample was inhibited by excess substrate (pyruvate). The mitoehondrial system showed NADH -cytochrome c oxidoreductase, succinate-cytochrome c oxidoreductase, NADH oxidase and some cytochrome c-oxygen oxidoreductase activities. Under anaerobic conditions, NADH-fumarate oxidoreductase and succinate-NAD + oxidoreductase activities of mitoehondrial preparations were stimulated in the presence of ADP and ATP respectively. Isolated mitochondria contained rhodoquinone and no ubiquinone, and isolated rhodoquinone was readily reduced by succinate in the presence of submitochondrial particles. Hydrogen peroxide was produced by submitochondrial particles in the presence or absence of KCN or in the presence of fumarate.     

Kinetic and structural analysis of Escherichia coli phosphoenolpyruvate carboxykinase mutants

BackgroundPhosphoenolpyruvate carboxykinase (PEPCK) is a metabolic enzyme in the gluconeogenesis pathway, where it catalyzes the reversible conversion of oxaloacetate (OAA) to phosphoenolpyruvate (PEP) and CO₂. The substrates for Escherichia coli PEPCK are OAA and MgATP, with Mn²⁺ acting as a cofactor. Analysis of PEPCK structures have revealed amino acid residues involved in substrate/cofactor coordination during catalysis.MethodsKey residues involved in coordinating the different substrates and cofactor bound to E. coli PEPCK were mutated. Purified mutant enzymes were used for kinetic assays. The structure of some mutant enzymes were determined using X-ray crystallography.ResultsMutation of residues D269 and H232, which comprise part of the coordination sphere of Mn²⁺, reduced k_cat by 14-fold, and significantly increased the K_m values for Mn²⁺ and OAA. Mutation of K254 a key residue in the P-loop motif that interacts with MgATP, significantly elevated the K_m value for MgATP and reduced k_cat. R65 and R333 are key residues that interacts with OAA. The R65Q and R333Q mutations significantly increased the K_m value for OAA and reduced k_cat respectively.ConclusionsOur results show that mutation of residues involved in coordinating OAA, MgATP and Mn²⁺ significantly reduce PEPCK activity. K254 plays an important role in phosphoryl transfer, while R333 is involved in both OAA decarboxylation and phosphoryl transfer by E. coli PEPCK.General significanceIn higher organisms including humans, PEPCK helps to regulate blood glucose levels, hence PEPCK is a potential drug target for patients with non-insulin dependent diabetes mellitus.