ABSTRACT
Hydroxamic acids derived from aldonic acids, namely aldonohydroxamic acids, have become an increasingly important class of inhibitors of enzymes involved in the metabolism of carbohydrates. We now report the straightforward preparation of various types of aldonohydroxamic acids by a new methodology involving the use of commercial 50% aqueous hydroxylamine as the source of the free base hydroxylamine that reacts directly with the corresponding aldonolactone dissolved in water. The reaction proceeds almost instantaneously in water at room temperature, yielding generally pure products in quantitative yield. To date, this methodology is probably the most facile and efficient way to synthesize aldonohydroxamic acids. We also determined by X-ray diffraction analysis the first crystal structure of a free aldonohydroxamic acid reported to date. Crystals of L-erythronohydroxamic acid belonged to the monoclinic system, space group P2(1), a=5.511(3), b=7.556(1), c=8.071(3) A, beta=109.10 degrees, and Z=2.
Subject(s)
Hydroxamic Acids/chemistry , Hydroxamic Acids/chemical synthesis , Hydroxylamine/chemistry , Lactones/chemistry , Water/chemistry , Crystallization , Hydroxamic Acids/analogs & derivatives , X-Ray Diffraction/methodsABSTRACT
Phosphoglucose isomerase (PGI; E.C. 5.3.1.9) catalyzes the second step in glycolysis, the interconversion of D-glucose-6-phosphate and D-fructose-6-phosphate. We determined the X-ray crystal structure of rabbit PGI complexed with a competitive inhibitor of isomerase activity, 5-phospho-D-arabinonate (5PAA), at 1.9 A resolution. 5PAA is a better mimic of the proposed cis-enediol(ate) intermediate than 6-phospho-D-gluconate, which was used in a previously reported crystal structure of rabbit PGI. The orientation of 5PAA bound in the enzyme active site predicts that active site residue Glu357 is the residue that transfers a proton between C2 and C1 of the proposed cis-enediol(ate) intermediate. Amino acid residues Arg272 and Lys210 are predicted to be involved in stabilizing the negative charge of the intermediate.
Subject(s)
Enzyme Inhibitors/chemistry , Glucose-6-Phosphate Isomerase/antagonists & inhibitors , Glucose-6-Phosphate Isomerase/chemistry , Glutamic Acid/chemistry , Pentosephosphates/chemistry , Animals , Binding, Competitive , Catalysis , Catalytic Domain , Crystallography, X-Ray , Enzyme Inhibitors/metabolism , Geobacillus stearothermophilus/enzymology , Glucose-6-Phosphate Isomerase/metabolism , Glutamic Acid/metabolism , Hydrogen-Ion Concentration , Muscle, Skeletal/enzymology , Pentosephosphates/metabolism , Phosphates/metabolism , Protein Binding , Protein Conformation , Rabbits , Structure-Activity Relationship , Substrate SpecificityABSTRACT
We report four new strong high energy intermediate analog competitive inhibitors of fructose-6-phosphate isomerization catalyzed by purified Trypanosoma brucei phosphoglucose isomerase: D-arabinonhydroxamic acid-5-phosphate, D-arabinonate-5-phosphate, D-arabinonamide-5-phosphate and D-arabinonhydrazide-5-phosphate. For comparison, the inhibitory properties of the corresponding non-phosphorylated analogues D-arabinonhydroxamic acid, D-arabinonate, D-arabinonamide and D-arabinonhydrazide were also evaluated. D-Arabinonhydroxamic acid-5-phosphate appears as the most potent competitive inhibitor ever evaluated on a phosphoglucose isomerase with an inhibition constant value of 50 nM and a Michaelis constant over inhibition constant ratio of about 2000. Our results show that anionic high energy intermediate analogues, and more particularly D-arabinonhydroxamic acid-5-phosphate, display a weak but significant specificity for Trypanosoma brucei phosphoglucose isomerase versus yeast phosphoglucose isomerase, while neutral high energy intermediate analogues are not selective at all. This would indicate the presence of more positively charged residues in the active site for Trypanosoma brucei phosphoglucose isomerase as compared to that of yeast phosphoglucose isomerase.
Subject(s)
Enzyme Inhibitors/pharmacology , Glucose-6-Phosphate Isomerase/antagonists & inhibitors , Pentosephosphates/pharmacology , Trypanosoma brucei brucei/enzymology , Animals , Binding, Competitive , Enzyme Inhibitors/chemical synthesis , Kinetics , Pentosephosphates/chemical synthesis , Protozoan Proteins/chemistry , Protozoan Proteins/metabolism , Trypanosoma brucei brucei/chemistryABSTRACT
The human oxytocinase/insulin-regulated aminopeptidase (OTase/IRAP) is a 1024 amino acid type II integral membrane protein that is expressed mainly in fat, muscle and placenta tissues. It has been thought to be involved mainly in the control of onset of labour but recently rat OTase/IRAP was shown to participate in the regulation of glucose transporter isoform 4 vesicle trafficking in adipocytes as well. To approach an understanding of OTase/IRAP gene regulation the organization of the human gene was determined. Accordingly, three overlapping genomic clones were isolated and characterized. The human OTase/IRAP gene (OTASE) was found to span approximately 75 kb containing 18 exons and 17 introns. The gluzincin aminopeptidase motif: GAMEN-(31 amino acids)-HELAH-(18 amino acids)-E associated with Zn2+-binding, substrate binding and catalysis is encoded by exons 6 and 7. A major and a minor transcriptional initiation site in OTASE were identified by primer extension 514 bp and 551 bp, respectively, upstream of the translation start codon. Chloroamphenicol acetyltransferase-reporter assays revealed a functional CpG-rich promoter/enhancer region located between nucleotide -621 and the major transcriptional initiation site. Human OTASE was assigned to chromosome 5 by hybridization to genomic DNA from characterized somatic cell hybrids. Finally, the OTASE and the human aminopeptidase A gene were subchromosomally localized to 5q21 and 4q25, respectively, by in situ hybridization.
Subject(s)
Aminopeptidases/genetics , Chromosomes, Human, Pair 5/genetics , Cystinyl Aminopeptidase/genetics , Amino Acid Sequence , Aminopeptidases/chemistry , Cell Line , Chromosome Mapping , Cloning, Molecular , Cystinyl Aminopeptidase/chemistry , Exons , Genes, Reporter , Humans , Hybrid Cells , In Situ Hybridization, Fluorescence , Introns , Molecular Sequence Data , Promoter Regions, Genetic , Sequence Alignment , TransfectionABSTRACT
Designed as competitive inhibitors of the isomerization reaction catalyzed by the potential chemotherapeutic target phosphoglucose isomerases (PGI), D-arabinonamide-5-phosphate and D-arabinohydrazine-5-phosphate were synthesized and fully characterized. These new types of phosphorylated sugar derivatives were easily and efficiently obtained in a one-step procedure from the promising synthon D-arabinono-1,4-lactone 5-phosphate. These two compounds proved to be new good competitive inhibitors of yeast PGI with the substrate D-fructose-6-phosphate, though not as strong as D-arabinohydroxamic acid-5-phosphate. Overall, our results are in accord with the postulated 1,2-cis-enediolate species as a probable high-energy intermediate of the PGI-catalyzed reaction.
Subject(s)
Enzyme Inhibitors/chemical synthesis , Glucose-6-Phosphate Isomerase/antagonists & inhibitors , Lactones/chemistry , Saccharomyces cerevisiae/enzymology , Sugar Phosphates/chemistry , Sugar Phosphates/chemical synthesis , Binding, Competitive , Drug Design , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Kinetics , Lactones/pharmacology , Structure-Activity Relationship , Sugar Phosphates/pharmacologyABSTRACT
Designed as a high energy intermediate analogue inhibitor of the potent chemotherapeutic target phosphoglucose isomerases, 5-phospho-D-arabinohydroxamate was efficiently synthesized in a two steps procedure. To date, it proved to be the strongest competitive inhibitor with respect to substrate D-fructose-6-phosphate (Ki down to 98 nM and Km/Ki values up to 513). A comparative inhibition study of this compound and other known strong inhibitors on phosphoglucose isomerases from three different sources is also reported.
