ADVERTISEMENT RETURN TO ISSUEPREVArticleOxygenation Mechanism of Ribulose-Bisphosphate Carboxylase/Oxygenase. Structure and Origin of 2-Carboxytetritol 1,4-Bisphosphate, a Novel O2-Dependent Side Product Generated by a Site-Directed MutantMark R. Harpel, Engin H. Serpersu, John A. Lamerdin, Zhi-Heng Huang, Douglas A. Gage, and Fred C. HartmanCite this: Biochemistry 1995, 34, 35, 11296–11306Publication Date (Print):September 5, 1995Publication History Published online1 May 2002Published inissue 5 September 1995https://pubs.acs.org/doi/10.1021/bi00035a039https://doi.org/10.1021/bi00035a039research-articleACS PublicationsRequest reuse permissionsArticle Views188Altmetric-Citations33LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-AlertscloseSupporting Info (3)»Supporting Information Supporting Information Get e-Alerts
Metabolic syndrome is a constellation of risk factors including hypertension, dyslipidemia, insulin resistance, and obesity that promote the development of cardiovascular disease. Metabolic syndrome has been associated with changes in the secretion or metabolism of glucocorticoids, which have important functions in adipose, liver, kidney, and vasculature. Tissue concentrations of the active glucocorticoid cortisol are controlled by the conversion of cortisone to cortisol by 11 β -hydroxysteroid dehydrogenase type 1 (11 β -HSD1). Because of the various cardiovascular and metabolic activities of glucocorticoids, we tested the hypothesis that 11 β -HSD1 is a common mechanism in the hypertension, dyslipidemia, and insulin resistance in metabolic syndrome. In obese and lean SHR/NDmcr-cp (SHR-cp), cardiovascular, metabolic, and renal functions were measured before and during four weeks of administration of vehicle or compound 11 (10 mg/kg/d), a selective inhibitor of 11 β -HSD1. Compound 11 significantly decreased 11 β -HSD1 activity in adipose tissue and liver of SHR-cp. In obese SHR-cp, compound 11 significantly decreased mean arterial pressure, glucose intolerance, insulin resistance, hypertriglyceridemia, and plasma renin activity with no effect on heart rate, body weight gain, or microalbuminuria. These results suggest that 11 β -HSD1 activity in liver and adipose tissue is a common mediator of hypertension, hypertriglyceridemia, glucose intolerance, and insulin resistance in metabolic syndrome.
Background The cardioprotective effects of glucagon-like peptide-1 (GLP-1) and analogs have been previously reported. We tested the hypothesis that albiglutide, a novel long half-life analog of GLP-1, may protect the heart against I/R injury by increasing carbohydrate utilization and improving cardiac energetic efficiency. Methods/Principal Findings Sprague-Dawley rats were treated with albiglutide and subjected to 30 min myocardial ischemia followed by 24 h reperfusion. Left ventricle infarct size, hemodynamics, function and energetics were determined. In addition, cardiac glucose disposal, carbohydrate metabolism and metabolic gene expression were assessed. Albiglutide significantly reduced infarct size and concomitantly improved post-ischemic hemodynamics, cardiac function and energetic parameters. Albiglutide markedly increased both in vivo and ex vivo cardiac glucose uptake while reducing lactate efflux. Analysis of metabolic substrate utilization directly in the heart showed that albiglutide increased the relative carbohydrate versus fat oxidation which in part was due to an increase in both glucose and lactate oxidation. Metabolic gene expression analysis indicated upregulation of key glucose metabolism genes in the non-ischemic myocardium by albiglutide. Conclusion/Significance Albiglutide reduced myocardial infarct size and improved cardiac function and energetics following myocardial I/R injury. The observed benefits were associated with enhanced myocardial glucose uptake and a shift toward a more energetically favorable substrate metabolism by increasing both glucose and lactate oxidation. These findings suggest that albiglutide may have direct therapeutic potential for improving cardiac energetics and function.
To explore the roles of active-site Glu" of ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum, the E48Q mutant has been characterized with respect to kinetics and product distribution.Although the kat for carboxylase activity is only 0.6% of the wild-type value, the mutant retains full activity in catalyzing the conversion of the carboxylated reaction intermediate to 3-phosphoglycerate and retains
Five residues (Thr-53, Asn-54, Gly-370, Gly-393, and Gly-394) of Rhodospirillum rubrum ribulose-bisphosphate carboxylase/oxygenase are positioned to serve as hydrogen-bond donors for the C1 phosphate of ribulose bisphosphate and thereby constrain conformational flexibility of the initial enediol(ate) intermediate (Knight, S., Andersson, I., and Branden, C.-I. (1990) J. Mol. Biol. 215, 113-160). To study the functional contributions of the residues implicated in ribulose bisphosphate binding and intermediate stabilization, we have replaced them individually with alanine, either to remove the H-bonding group (T53A, N54A) or to introduce bulk (G370A, G393A, G394A). Consequences of substitutions include diminution of carboxylase activity (with a lesser impact on enolization activity), increase of Km (ribulose bisphosphate), and decrease of carboxylation: oxygenation specificity. During catalytic turnover of ribulose bisphosphate by several mutants, substantial amounts of the substrate are diverted to 1-deoxy-D-glycero-2,3-pentodiulose 5-phosphate, reflecting beta-elimination of phosphate from the enediol(ate) intermediate. This side product is not observed with wild-type enzyme, nor has it been reported with mutant enzymes characterized previously. Another consequence of disruption of the phosphate binding site is enhanced production of pyruvate, relative to wild-type enzyme, by some of the mutants due to decomposition of the acicarbanion of 3-phosphoglycerate (the terminal intermediate). These data provide direct evidence that phosphate ligands stabilize conformations of intermediates that favor productive turnover and mitigate beta-elimination at two stages of overall catalysis.
Antithrombotic agents that are inhibitors of factor XIa (FXIa) have the potential to demonstrate robust efficacy with a low bleeding risk profile. Herein, we describe a series of tetrahydroquinoline (THQ) derivatives as FXIa inhibitors. Compound 1 was identified as a potent and selective tool compound for proof of concept studies. It exhibited excellent antithrombotic efficacy in rabbit thrombosis models and did not prolong bleeding times. This demonstrates proof of concept for the FXIa mechanism in animal models with a reversible, small molecule inhibitor.
Abstract Given the current state of knowledge of the reaction pathways catalysed by Rubisco* (Jaworowski and Rose 1985; Pierce et al. 1986a; Schloss 1990) and the elucidation of the three-dimensional structure of several different forms of the enzyme (Chapman et al. 1988; Andersson et al. 1989; Knight et al. 1990; Schneider et al. 1990; Lundqvist and Schneider 1991), site-directed mutagenesis offers the potential to decipher catalytic roles of active-site residues and to unravel the functional significance of various structural elements. Especially intriguing are intersubunit, electrostatic interactions at the active site between Glu48+ and Lys168 of the nonactivated (noncarbamylated) enzyme (Schneider et al. 1990) and between Glu48 and Lys329 of the activated (carbamylated) enzyme (Knight et al.1990).
The nonheme iron oxidase isopenicillin N synthase catalyzes the formation of two new internal bonds in the tripeptide δ-(L-α-aminoadipoyl)-L-cysteinyl-D-valine (ACV) to form the β-lactam and thiazolidine rings of isopenicillin N. Concomitantly, O2 is reduced to 2 H2O. The recombinant enzyme from Cephalosporium acremonium (Mr = 38,400), expressed as an apoenzyme in Escherichia coli, binds 1 g atom of Fe2+/mol of enzyme to reconstitute full activity. Mossbauer spectra of the 57Fe-enriched enzyme exhibit parameters (δ = 1.30 mm/s, Δ EQ = 2.70 mm/s) which unambiguously show that the active site iron is high spin Fe2+. Anaerobic binding of ACV causes a substantial decrease in the isomer shift parameter δ (δ = 1.10 mm/s, ΔEQ = 3.40 mm/s) showing that the substrate perturbs the iron site and makes its coordination environment much more covalent. Nitric oxide (NO) binds to the EPR silent active site iron to give an EPR active species (g = 4.09, 3.95, 2.0; S = 3/2) similar to those of the nitrosyl complexes of many other mononuclear Fe2+-containing enzymes. The rhombicity of the EPR spectrum is increased (g = 4.22, 3.81, 1.99) by anaerobic addition of ACV suggesting that the substrate binds to or near the iron without displacing NO. Interestingly, the enzyme·ACV·NO complex displays an optical spectrum similar to that of ferric rubredoxin in which the iron has only thiol coordination. This suggests that the Fe2+ of the enzyme· ACV·NO complex acquires Fe3+ character and that the cysteinyl thiol moiety of ACV coordinates to the iron. Similar substrate thiol coordination to the iron of the enzyme·ACV complex is the most probable explanation for the large decrease in isomer shift observed. These results provide the first evidence for the direct involvement of iron in this unique O2-dependent reaction and suggest novel roles for iron and oxygen in biological catalysis.
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