RESUMEN

Glycogen phosphorylases catalyze the breakdown of glycogen to glucose-1-phosphate, which enters glycolysis to fulfill the energetic requirements of the organism. Maintaining control of blood glucose levels is critical in minimizing the debilitating effects of diabetes, making liver glycogen phosphorylase a potential therapeutic target. To support inhibitor design, we determined the crystal structures of the active and inactive forms of human liver glycogen phosphorylase a. During activation, forty residues of the catalytic site undergo order/disorder transitions, changes in secondary structure, or packing to reorganize the catalytic site for substrate binding and catalysis. Knowing the inactive and active conformations of the liver enzyme and how each differs from its counterpart in muscle phosphorylase provides the basis for designing inhibitors that bind preferentially to the inactive conformation of the liver isozyme.

Asunto(s)

Hígado/enzimología , Fosforilasas/química , Fosforilasas/metabolismo , Adenosina Monofosfato/metabolismo , Animales , Sitios de Unión , Dominio Catalítico , Cristalografía por Rayos X , Diabetes Mellitus/tratamiento farmacológico , Diabetes Mellitus/metabolismo , Diseño de Fármacos , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Humanos , Técnicas In Vitro , Modelos Moleculares , Músculos/enzimología , Fosforilasas/genética , Conformación Proteica , Estructura Secundaria de Proteína , Conejos

RESUMEN

Squalene synthase catalyzes the biosynthesis of squalene, a key cholesterol precursor, through a reductive dimerization of two farnesyl diphosphate (FPP) molecules. The reaction is unique when compared with those of other FPP-utilizing enzymes and proceeds in two distinct steps, both of which involve the formation of carbocationic reaction intermediates. Because FPP is located at the final branch point in the isoprenoid biosynthesis pathway, its conversion to squalene through the action of squalene synthase represents the first committed step in the formation of cholesterol, making it an attractive target for therapeutic intervention. We have determined, for the first time, the crystal structures of recombinant human squalene synthase complexed with several different inhibitors. The structure shows that SQS is folded as a single domain, with a large channel in the middle of one face. The active sites of the two half-reactions catalyzed by the enzyme are located in the central channel, which is lined on both sides by conserved aspartate and arginine residues, which are known from mutagenesis experiments to be involved in FPP binding. One end of this channel is exposed to solvent, whereas the other end leads to a completely enclosed pocket surrounded by conserved hydrophobic residues. These observations, along with mutagenesis data identifying residues that affect substrate binding and activity, suggest that two molecules of FPP bind at one end of the channel, where the active center of the first half-reaction is located, and then the stable reaction intermediate moves into the deep pocket, where it is sequestered from solvent and the second half-reaction occurs. Five alpha helices surrounding the active center are structurally homologous to the active core in the three other isoprenoid biosynthetic enzymes whose crystal structures are known, even though there is no detectable sequence homology.

Asunto(s)

Farnesil Difosfato Farnesil Transferasa/química , Transferasas Alquil y Aril/química , Secuencia de Aminoácidos , Sitios de Unión , Dominio Catalítico , Colesterol/biosíntesis , Simulación por Computador , Secuencia Conservada , Cristalografía por Rayos X , Farnesil Difosfato Farnesil Transferasa/antagonistas & inhibidores , Geranilgeranil-Difosfato Geranilgeraniltransferasa , Geraniltranstransferasa , Humanos , Liasas Intramoleculares/química , Modelos Químicos , Modelos Moleculares , Familia de Multigenes , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido

RESUMEN

A new class of glutathione transferases has been discovered by analysis of the expressed sequence tag data base and sequence alignment. Glutathione S-transferases (GSTs) of the new class, named Omega, exist in several mammalian species and Caenorhabditis elegans. In humans, GSTO 1-1 is expressed in most tissues and exhibits glutathione-dependent thiol transferase and dehydroascorbate reductase activities characteristic of the glutaredoxins. The structure of GSTO 1-1 has been determined at 2.0-A resolution and has a characteristic GST fold (Protein Data Bank entry code ). The Omega class GSTs exhibit an unusual N-terminal extension that abuts the C terminus to form a novel structural unit. Unlike other mammalian GSTs, GSTO 1-1 appears to have an active site cysteine that can form a disulfide bond with glutathione.

Asunto(s)

Glutatión Transferasa/química , Glutatión Transferasa/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Caenorhabditis elegans/enzimología , Cristalografía por Rayos X , Femenino , Glutatión Transferasa/genética , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Masculino , Mamíferos , Modelos Moleculares , Datos de Secuencia Molecular , Filogenia , Conformación Proteica , Estructura Secundaria de Proteína , Lugares Marcados de Secuencia , Especificidad por Sustrato , Transcripción Genética

RESUMEN

Through the use of empirical and computational methods, phosphinate-based inhibitors of MMP-1 and MMP-13 that bind into the S2 pocket of these enzymes were designed. The synthesis and testing of 2 suggested that binding was occurring as hypothesized. Structure determination of a co-crystal of 2 bound to the catalytic domain of MMP-1 confirmed the binding mode. Substituents binding into S2, S1', S2' and S3', were optimized yielding compounds with low double-digit nM IC50's against these enzymes.

Asunto(s)

Inhibidores de la Metaloproteinasa de la Matriz , Ácidos Fosfínicos/farmacología , Sitios de Unión , Colagenasas/farmacocinética , Simulación por Computador , Cristalografía por Rayos X , Diseño de Fármacos , Concentración 50 Inhibidora , Metaloproteinasa 1 de la Matriz , Metaloproteinasa 13 de la Matriz , Modelos Moleculares

Asunto(s)

Fructosa/análogos & derivados , Cristalografía por Rayos X , Fructosa/química , Modelos Moleculares , Conformación Molecular

RESUMEN

Chemical analysis of the eggs spawned by fiveSinularia soft corals (Octocorallia, Alcyoniidae) collected in the central region of the Great Barrier Reef, Australia, revealed the presence of the known cembranoid diterpenes epoxypukalide and/or pukalide in all cases. Examination of the colonies from which the eggs were released one month before spawning and also some time after spawning failed to detect either diterpene in the coral tissue. The three dimensional structures of pukalide and epoxypukalide, as determined by the single-crystal X-ray technique, are reported for the first time. The wide distribution of pukalide and epoxypukalide and of related compounds within the octocorallia is discussed.