Mammalian stearoyl-CoA desaturase-1 (SCD1) introduces a double-bond to a saturated long-chain fat... more Mammalian stearoyl-CoA desaturase-1 (SCD1) introduces a double-bond to a saturated long-chain fatty acid and the reaction is catalyzed by a diiron center, which is well-coordinated by conserved histidine residues and is thought to remain with enzyme. However, we find that SCD1 progressively loses its activity during catalysis and becomes fully inactive after nine turnovers. Further studies show that the inactivation of SCD1 is due to the loss of an iron (Fe) ion in the diiron center, and that the addition of free ferrous ions (Fe2+) sustains the enzymatic activity. Using SCD1 labeled with Fe isotope, we further show that free Fe2+is incorporated into the diiron center only during catalysis. We also discover that the diiron center in SCD1 has prominent electron paramagnetic resonance signals in its diferric state, indicative of distinct coupling between the two ferric ions. These results reveal that the diiron center in SCD1 is structurally dynamic during catalysis and that labile Fe...
The pH- and temperature-dependent changes in the coordination sphere of the heme c group of N alp... more The pH- and temperature-dependent changes in the coordination sphere of the heme c group of N alpha-acetyl microperoxidase-8 (Ac-MP-8) have been studied by examining its optical, resonance Raman, electron paramagnetic resonance, and magnetic circular dichroism spectra. An optical titration indicates that Ac-MP-8 exists in three major ionization forms over the pH 1-12 range that are linked by pK alpha values of approximately 3 and 9. The acid form that is present at pH 1.5 exists as a mixture of five- and six-coordinate high-spin species and most likely has water or buffer ions as axial ligand(s). On titration to pH 7, the His18 residue is deprotonated and becomes the proximal ligand to the iron to give a six-coordinate neutral form that has water as the sixth ligand. This form exists in a thermal high-spin intermediate-spin state equilibrium. On raising the pH to 10, an alkaline form is generated which is predominantly a five-coordinate high-spin species. It is formed by ionization of the proximal His18 residue to its imidazolate form with concomitant dissociation of the water ligand at the sixth site. At concentrations of Ac-MP-8 greater than 10 microM, some six-coordinate low-spin species are formed that are attributed to a dimer in which a His18 residue from a second molecule of Ac-MP-8 coordinates to the sixth site of another to give a bis-His complex. Raising the pH to 11.5 does not produce an appreciable amount of the six-coordinate complex with hydroxide as the sixth ligand. These studies show that Ac-MP-8 is a good water-soluble model for the peroxidases that exhibits minimal aggregation at concentrations below 10 microM in the neutral and alkaline pH regions.
ABSTRACT Prostaglandin H (PGH) synthase (EC 1.14.99.1) is a key enzyme in the biosynthesis of pro... more ABSTRACT Prostaglandin H (PGH) synthase (EC 1.14.99.1) is a key enzyme in the biosynthesis of prostaglandins, thromboxane, and prostacyclin. In cultured human umbilical vein endothelial cells, interleukin 1 (IL-1) is known to induce the synthesis of this enzyme, thereby raising the level of PGH synthase protein severalfold over the basal level. Pretreatment with aspirin at low concentrations (0.1-1 micrograms/ml) inhibited more than 60% of the enzyme mass and also the cyclooxygenase activity in IL-1-induced cells with only minimal effects on the basal level of the synthase enzyme in cells without IL-1. Sodium salicylate exhibited a similar inhibitory action whereas indomethacin had no apparent effect. Similarly low levels of aspirin inhibited the increased L-[35S]methionine incorporation into PGH synthase that was induced by IL-1 and also suppressed expression of the 2.7-kilobase PGH synthase mRNA. These results suggest that in cultured endothelial cells a potent inhibition of eicosanoid biosynthetic capacity can be effected by aspirin or salicylate at the level of PGH synthase gene expression. The aspirin effect may well be due to degradation of salicylate.
Mammalian stearoyl-CoA desaturase-1 (SCD1) introduces a double-bond to a saturated long-chain fat... more Mammalian stearoyl-CoA desaturase-1 (SCD1) introduces a double-bond to a saturated long-chain fatty acid and the reaction is catalyzed by a diiron center, which is well-coordinated by conserved histidine residues and is thought to remain with enzyme. However, we find that SCD1 progressively loses its activity during catalysis and becomes fully inactive after nine turnovers. Further studies show that the inactivation of SCD1 is due to the loss of an iron (Fe) ion in the diiron center, and that the addition of free ferrous ions (Fe2+) sustains the enzymatic activity. Using SCD1 labeled with Fe isotope, we further show that free Fe2+is incorporated into the diiron center only during catalysis. We also discover that the diiron center in SCD1 has prominent electron paramagnetic resonance signals in its diferric state, indicative of distinct coupling between the two ferric ions. These results reveal that the diiron center in SCD1 is structurally dynamic during catalysis and that labile Fe...
The pH- and temperature-dependent changes in the coordination sphere of the heme c group of N alp... more The pH- and temperature-dependent changes in the coordination sphere of the heme c group of N alpha-acetyl microperoxidase-8 (Ac-MP-8) have been studied by examining its optical, resonance Raman, electron paramagnetic resonance, and magnetic circular dichroism spectra. An optical titration indicates that Ac-MP-8 exists in three major ionization forms over the pH 1-12 range that are linked by pK alpha values of approximately 3 and 9. The acid form that is present at pH 1.5 exists as a mixture of five- and six-coordinate high-spin species and most likely has water or buffer ions as axial ligand(s). On titration to pH 7, the His18 residue is deprotonated and becomes the proximal ligand to the iron to give a six-coordinate neutral form that has water as the sixth ligand. This form exists in a thermal high-spin intermediate-spin state equilibrium. On raising the pH to 10, an alkaline form is generated which is predominantly a five-coordinate high-spin species. It is formed by ionization of the proximal His18 residue to its imidazolate form with concomitant dissociation of the water ligand at the sixth site. At concentrations of Ac-MP-8 greater than 10 microM, some six-coordinate low-spin species are formed that are attributed to a dimer in which a His18 residue from a second molecule of Ac-MP-8 coordinates to the sixth site of another to give a bis-His complex. Raising the pH to 11.5 does not produce an appreciable amount of the six-coordinate complex with hydroxide as the sixth ligand. These studies show that Ac-MP-8 is a good water-soluble model for the peroxidases that exhibits minimal aggregation at concentrations below 10 microM in the neutral and alkaline pH regions.
ABSTRACT Prostaglandin H (PGH) synthase (EC 1.14.99.1) is a key enzyme in the biosynthesis of pro... more ABSTRACT Prostaglandin H (PGH) synthase (EC 1.14.99.1) is a key enzyme in the biosynthesis of prostaglandins, thromboxane, and prostacyclin. In cultured human umbilical vein endothelial cells, interleukin 1 (IL-1) is known to induce the synthesis of this enzyme, thereby raising the level of PGH synthase protein severalfold over the basal level. Pretreatment with aspirin at low concentrations (0.1-1 micrograms/ml) inhibited more than 60% of the enzyme mass and also the cyclooxygenase activity in IL-1-induced cells with only minimal effects on the basal level of the synthase enzyme in cells without IL-1. Sodium salicylate exhibited a similar inhibitory action whereas indomethacin had no apparent effect. Similarly low levels of aspirin inhibited the increased L-[35S]methionine incorporation into PGH synthase that was induced by IL-1 and also suppressed expression of the 2.7-kilobase PGH synthase mRNA. These results suggest that in cultured endothelial cells a potent inhibition of eicosanoid biosynthetic capacity can be effected by aspirin or salicylate at the level of PGH synthase gene expression. The aspirin effect may well be due to degradation of salicylate.
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Papers by Ah-lim Tsai