4 resultados para Catalytic Mechanism
em BORIS: Bern Open Repository and Information System - Berna - Suiça
Resumo:
Apparent mineralocorticoid excess (AME) is a severe form of hypertension that is caused by impaired activity of 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2), which converts biologically active cortisol into inactive cortisone. Mutations in HSD11B2 result in cortisol-induced activation of mineralocorticoid receptors and cause hypertension with hypokalemia, metabolic alkalosis, and suppressed circulating renin and aldosterone concentrations. This study uncovered the first patient with AME who was described in the literature, identified the genetic defect in HSD11B2, and provided evidence for a novel mechanism of reduced 11beta-HSD2 activity. This study identified a cluster of amino acids (335 to 339) in the C-terminus of 11beta-HSD2 that are essential for protein stability. The cluster includes Tyr(338), which is mutated in the index patient, and Arg(335) and Arg(337), previously reported to be mutated in hypertensive patients. It was found that wild-type 11beta-HSD2 is a relatively stable enzyme with a half-life of 21 h, whereas that of Tyr(338)His and Arg(337)His was 3 and 4 h, respectively. Enzymatic activity of Tyr(338)His was partially retained at 26 degrees C or in the presence of the chemical chaperones glycerol and dexamethasone, indicating thermodynamic instability and misfolding. The results provide evidence that the degradation of both misfolded mutant Tyr(338)His and wild-type 11beta-HSD2 occurs through the proteasome pathway. Therefore, impaired 11beta-HSD2 protein stability rather than reduced gene expression or loss of catalytic activity seems to be responsible for the development of hypertension in some individuals with AME.
Resumo:
The haem detoxification pathway of the malaria parasite Plasmodium falciparum is a potential biochemical target for drug development. Free haem, released after haemoglobin degradation, is polymerized by the parasite to form haemozoin pigment. Plasmodium falciparum histidine-rich protein-2 (Pfhrp-2) has been implicated as the catalytic scaffold for detoxification of haem in the malaria parasite. Previously we have shown that a hexapeptide repeat sequence (Ala-His-His-Ala-Ala-Asp), which appears 33 times in Pfhrp-2, may be the major haem binding site in this protein. The haem binding studies carried out by ourselves indicate that up to 18 equivalents of haem could be bound by this protein with an observed K(d) of 0.94 microM. Absorbance spectroscopy provides evidence that chloroquine is capable of extracting haem bound to Pfhrp-2. This was supported by the K(d) value, of 37 nM, observed for the haem-chloroquine complex. The native PAGE studies reveal that the formation of the haem-Pfhrp-2 complex is disrupted by chloroquine. These results indicate that chloroquine may be acting by inhibiting haem detoxification/binding to Pfhrp-2. Moreover, the higher affinity of chloroquine for haem than Pfhrp-2 suggests a possible mechanism of action for chloroquine; it may remove the haem bound to Pfhrp-2 and form a complex that is toxic to the parasite.
Resumo:
DNAzymes (Dz) 8–17 and 10–23 are two widely studied and well-characterized RNA-cleaving DNA catalysts. In an effort to further improve the understanding of the fragile interactions and dynamics of the enzymatic mechanism, this study examines the catalytic efficiency of minimally modified DNAzymes. Five single mutants of Dz8–17 and Dz10–23 were prepared by replacing the adenine residues in the corresponding catalytic cores with 3-deazaadenine units. Kinetic assays were used to assess the effect on the catalytic activity and thereby identify the importance of hydrogen bonding that arises from the N3 atoms. The results suggest that modifications at A15 and A15.0 of Dz8–17 have a significant influence and show a reduction in catalytic activity. Modification at each location in Dz10–23 results in a decrease of the observed rate constants, with A12 appearing to be the most affected with a reduction of ∼80% of kobs and ∼25% of the maximal cleavage rate compared to the wild-type DNAzyme. On the other hand, modification of A12 in Dz8–17 showed an ∼130% increase in kobs, thus unraveling a new potential site for the introduction of chemical modifications. A pH-profile analysis showed that the chemical cleavage step is rate-determining, regardless of the presence and/or location of the mutation. These findings point towards the importance of the N3-nitrogens of certain adenine nucleotides located within the catalytic cores of the DNAzymes for efficient catalytic activity and further suggest that they might directly partake in maintaining the appropriate tertiary structure. Therefore, it appears that minor groove interactions constitute an important feature of DNAzymes as well as ribozymes.