RESUMEN
Squalene synthase catalyzes two consecutive reactions in sterol biosynthesis-the condensation of two molecules of farnesyl diphosphate (FPP) to form the cyclopropylcarbinyl intermediate presqualene diphosphate (PSPP) and the subsequent rearrangement and reduction of PSPP to form squalene. Steady-state and pre-steady-state kinetic studies, in combination with isotope-trapping experiments of enzyme.substrate complexes, indicate that two molecules of FPP add to the enzyme before NADPH and that PSPP is converted directly to squalene without dissociating from the enzyme under normal catalytic conditions. In addition, formation of PSPP or a prior conformational change in squalene synthase is the rate-limiting step for synthesis of squalene from FPP via PSPP in the presence of NADPH and for synthesis of PSPP in the absence of NADPH. Squalene synthase is inhibited at high concentrations of FPP. Inhibition is specific for the formation of squalene, but not PSPP, and is competitive with respect to NADPH. In addition, the binding of either NADPH or a third, nonreacting molecule of FPP stimulates the rate of PSPP formation. A kinetic mechanism is proposed to account for these observations.
Asunto(s)
Farnesil Difosfato Farnesil Transferasa/química , Farnesil Difosfato Farnesil Transferasa/metabolismo , Unión Competitiva , Radioisótopos de Carbono , Hidrógeno/química , Cinética , Modelos Químicos , NADP/química , NADP/metabolismo , Fosfatos de Poliisoprenilo/química , Fosfatos de Poliisoprenilo/metabolismo , Unión Proteica , Saccharomyces cerevisiae/enzimología , Sesquiterpenos , Escualeno/antagonistas & inhibidores , Escualeno/metabolismo , Especificidad por Sustrato , TritioRESUMEN
Farnesyl protein transferase (FPT) is an alpha/beta heterodimeric zinc enzyme that catalyzes posttranslational farnesylation of many key cellular regulatory proteins, including oncogenic Ras. On the basis of the recently reported crystal structure of FPT complexed with a CVIM peptide and alpha-hydroxyfarnesylphosphonic acid, site-directed mutagenesis of the FPT active site was performed so key residues that are responsible for substrate binding and catalysis could be identified. Eight single mutants, including K164N alpha, Y166F alpha, Y166A alpha, Y200F alpha, H201A alpha, H248A beta, Y300F beta, and Y361F beta, and a double mutant, H248A beta/Y300F beta, were prepared. Steady-state kinetic analysis along with structural evidence indicated that residues Y200 alpha, H201 alpha, H248 beta, and Y361 beta are mainly involved in substrate binding. In addition, biochemical results confirm structural observations which show that residue Y166 alpha plays a key role in stabilizing the active site conformation of several FPT residues through cation-pi interactions. Two mutants, K164N alpha and Y300F beta, have moderately decreased catalytic constants (kcat). Pre-steady-state kinetic analysis of these mutants from rapid quench experiments showed that the chemical step rate constant was reduced by 41- and 30-fold, respectively. The product-releasing rate for each dropped approximately 10-fold. In pH-dependent kinetic studies, Y300F beta was observed to have both acidic and basic pKa values shifted 1 log unit from those of the wild-type enzyme, consistent with a possible role for Y300 beta as an acid-base catalyst. K164N alpha had a pKa shift from 6.0 to 5.3, which suggests it may function as a general acid. On the basis of these results along with structural evidence, a possible FPT reaction mechanism is proposed with both Y300 beta and K164 alpha playing key catalytic roles in enhancing the reactivity of the farnesyl diphosphate leaving group.
Asunto(s)
Transferasas Alquil y Aril/genética , Transferasas Alquil y Aril/metabolismo , Lisina/genética , Lisina/metabolismo , Tirosina/genética , Tirosina/metabolismo , Transferasas Alquil y Aril/química , Animales , Sitios de Unión/genética , Catálisis , Concentración de Iones de Hidrógeno , Cinética , Lisina/química , Modelos Químicos , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Prenilación de Proteína/genética , Ratas , Tirosina/químicaRESUMEN
The gene responsible for hereditary hemochromatosis (HH) is tightly linked to the class I region of the human leukocyte antigen (HLA) complex. Initial studies designed to map the disease locus have relied on serological markers for the class I antigens. Molecular markers from this region can now be used in combination with HLA serotyping for mapping studies. We previously reported two pedigrees in which serological data indicated recombinant events within the class I region. These data suggested a location for the HH locus between HLA-A and HLA-B. Molecular mapping studies have allowed us to demonstrate that an apparent recombination in one pedigree did not occur. This approach has also produced a more precise centromeric boundary for the region containing the disease locus, telomeric of HLA-C. These results emphasize the importance of including both serological and molecular markers in pedigree studies aimed at fine mapping the HH locus.
Asunto(s)
Marcadores Genéticos , Hemocromatosis/genética , Recombinación Genética , Células Cultivadas , Mapeo Cromosómico , Femenino , Ligamiento Genético , Antígenos HLA/genética , Humanos , Masculino , Linaje , Polimorfismo de Longitud del Fragmento de RestricciónRESUMEN
Uroporphyrinogen decarboxylase (URO-D) is a cytosolic heme-biosynthetic enzyme that converts uroporphyrinogen to coproporphyrinogen. Defects at the uroporphyrinogen decarboxylase locus cause the human genetic disease familial porphyria cutanea tarda. A splice site mutation has been found in a pedigree with familial porphyria cutanea tarda that causes exon 6 to be deleted from the mRNA. The intron/exon junctions on either side of exon 6 fall between codons, so the resulting protein is shorter than the normal protein, missing only the amino acids coded by exon 6. The shortened protein lacks catalytic activity, is rapidly degraded when exposed to human lymphocyte lysates, and is not detectable by Western blot analysis in lymphocyte lysates derived from affected individuals. The mutation was detected in five of 22 unrelated familial porphyria cutanea tarda pedigrees tested, so it appears to be common. This is the first splice site mutation to be found at the URO-D locus, and the first mutation that causes familial porphyria cutanea tarda to be found in more than one pedigree.