ABSTRACT
The first chemical synthesis of D-neopterin-3'-triphosphate and D-7,8-dihydroneopterin-3'-triphosphate is described. D-neopterin-3'-monophosphate was first 1'-2'-0-formylated with anhydrous formic acid, then activated with 1,1'-carbonyldiimidazole and phosphorylated with n-tributyl-ammonium pyrophosphate. The yield of 3'-NTP was 24%. D-7,8-dihydroneopterin-3'-triphosphate was obtained by chemical (hyposulfite) or catalytic (Pd:H2) reduction of 3'-NTP. Preparations from both reductions were fully active in two different enzymatic systems: synthesis of L-5,6,7,8-tetrahydrobiopterin and in the C-2'-epimerization reaction to L-7,8-dihydromonapterin-3'-triphosphate.
Subject(s)
Neopterin , Pteridines/chemical synthesis , Biopterins/analogs & derivatives , Biopterins/chemical synthesis , MethodsABSTRACT
An enzyme catalyzing the elimination of triphosphate from 7,8-dihydroneopterin triphosphate in the presence of Mg2+ has been purified approx. 3000 fold from human liver. It has a molecular weight of approx. 63'000, a pI value of 4.4 - 4.6 and is stable at 80 degrees C for 5 min. This enzyme catalyzes the formation of tetrahydrobiopterin in the presence of sepiapterin reductase, Mg2+ and NADPH. It is thus possible, that it also catalyzes the internal oxidoreduction leading to formation of the intermediate 6-pyruvoyl-tetrahydropterin, suggesting that no further enzyme is obligatory for biosynthesis of tetrahydrobiopterin.
Subject(s)
Biopterins/biosynthesis , Liver/analysis , Pteridines/biosynthesis , Pyrophosphatases/isolation & purification , Biopterins/analogs & derivatives , Humans , Isoelectric Point , Magnesium/metabolism , Molecular Weight , NADP/metabolism , Neopterin/analogs & derivatives , Pteridines/metabolism , Pyrophosphatases/metabolism , Temperature , Time FactorsABSTRACT
A critical review on corrinoid biochemistry and physiology is presented. This includes: chemical synthesis of biologically important organocorrinoids and the correlation between their structures and coenzymatic activity; forms, distribution and transport of physiologically active corrinoids; methylcobalamin- and adenosylcobalamin-dependent enzymatic reactions and their physiological functions; and steric course of the adenosylcobalamin-dependent enzymatic rearrangements. Special attention is paid to the mechanisms of adenosylcobalamin-dependent enzymatic reactions.
Subject(s)
Vitamin B 12/metabolism , Cobamides/metabolism , Cobamides/pharmacology , Enzymes/metabolism , Models, Biological , Structure-Activity Relationship , Vitamin B 12/analogs & derivatives , Vitamin B 12/chemical synthesis , Vitamin B 12/pharmacologyABSTRACT
The serum extracts were purified by column-, thin layer- and high pressure liquid chromatography. Deuterated cholecalciferol and deuterated 25-hydroxycholecalciferol were used in internal standards. The quantitative analysis was performed using GC-mass fragmentography technique of TMS-ethers.
Subject(s)
Cholecalciferol/blood , Hydroxycholecalciferols/blood , Gas Chromatography-Mass Spectrometry , Humans , MethodsABSTRACT
1. Ethylmalonyl-CoA was found to be a substrate for methylmalonyl-CoA mutase from Propionibacterium shermanii, the product being mainly (2R)-methylsuccinyl-CoA along with some (2S)-diastereoisomer. 2. The relevant 1H-nuclear magnetic resonance signals of methylsuccinic acid and of its dimethyl ester were assigned to the diastereotopic methylene hydrogens using sterospecifically dideuterated specimens of known configuration. 3. [2(-2)H1]Ethylmalonyl-CoA was converted by methylmalonyl-CoA mutase in 2H2O mainly to (2R, 3S)-[3(-2)H1]methylsuccinyl-CoA. No dideuterated product was observed. 4. Starting from (1R)-[1(-2)H1]-ethathanol, (1S)-[1(-2)H1]ethanol and [2H6] ethanol the following deuterated specimens of ethylmalonic acid were synthesised and characterised: (3S)-[3(-2)H1], (3R)-[3(-2)H1] and [3(-2)H2, 4(-2)H3], respectively. 5. Conversion of (3S)-[3(-2)H1]-ethylmalonyl-CoA (70% 2H1 and 2% 2H2 species) on the mutase in water afforded mainly (2R)-[2(-2)H1]methylsuccinyl-CoA along with some (2S)-diastereoisomer. No deuterium loss was observed. 6. Methylmalonyl-CoA mutase converted (3R)-[3(-2)H1]ethylmalonyl-CoA (81% 2H1 and 2% 2H2 species) to the following methylsuccinyl-CoA species: 33% [3(-2)H1], the deuterium being in the threo position with respect to the methyl group; 21% [2(-2)H1]; 46% unlabelled. The ratio of the species with (2R) and (2S) configuration was about 60:40. 7. Reaction of [3(-2)H2, 4(-2)H3]ethylmalonyl-CoA (94.5% [2H5] species) with the mutase gave the following labelled methylsuccinyl-CoA species:53.4% [methyl-2H3, 2(-2)H1, 3(-2)H1], the 3-deuterium being in the threo position with respect to the methyl group; 37.6% [methyl-2H3, 2(-2)H1]; 5% [methyl(-2)H3, 2(-2)H1, 2(-2)H1, 3(-2)H1] the 3-deuterium being in erythro position with respect to the methyl group; 4% [methyl(-2)H3, 3(-2)H1]. The ratio of the species with (2R) and (2S) configuration was about 70:30. 8. Implications of these findings for the mechanism of the rearrangements catalysed by coenzyme B12 are discussed.
