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Transition-state Analogue (transition-state + analogue)
Selected AbstractsCrystallographic binding studies with an engineered monomeric variant of triosephosphate isomeraseACTA CRYSTALLOGRAPHICA SECTION D, Issue 8 2010Mikko Salin Crystallographic binding studies have been carried out to probe the active-site binding properties of a monomeric variant (A-TIM) of triosephosphate isomerase (TIM). These binding studies are part of a structure-based directed-evolution project aimed towards changing the substrate specificity of monomeric TIM and are therefore aimed at finding binders which are substrate-like molecules. A-TIM has a modified more extended binding pocket between loop-7 and loop-8 compared with wild-type TIM. The A-TIM crystals were grown in the presence of citrate, which is bound in the active site of each of the two molecules in the asymmetric unit. In this complex, the active-site loops loop-6 and loop-7 adopt the closed conformation, similar to that observed in liganded wild-type TIM. Extensive crystal-soaking protocols have been developed to flush the bound citrate out of the active-site pocket of both molecules and the crystal structure shows that the unliganded open conformation of the A-TIM active site is the same as in unliganded wild-type TIM. It is also shown that sulfonate compounds corresponding to the transition-state analogue 2-phosphoglycolate bind in the active site, which has a closed conformation. It is also shown that the new binding pocket of A-TIM can bind 3-phosphoglycerate (3PGA; an analogue of a C4-sugar phosphate) and 4-phospho- d -erythronohydroxamic acid (4PEH; an analogue of a C5-sugar phosphate). Therefore, these studies have provided a rationale for starting directed-evolution experiments aimed at generating the catalytic properties of a C5-sugar phosphate isomerase on the A-TIM framework. [source] Crystallization of a carbamatase catalytic antibody Fab fragment and its complex with a transition-state analogueACTA CRYSTALLOGRAPHICA SECTION D, Issue 1 2004Carbamatase catalytic antibody Fab fragment Catalytic antibodies showing carbamatase activity have significant potential in antibody-directed prodrug therapy against tumours. The Fab fragment of an IgG1 mouse monoclonal carbamatase catalytic antibody JC1 raised against a transition-state analogue, ethyl N -(3,5-dicarboxyphenyl)- P -{N- [5,-(2,,,5,,-dioxo-1,,-pyrrolidinyl)oxy-1,,5,-dioxopentyl]-4-aminophenylmethyl}phosphonamidate, was obtained by digestion of the whole antibody with papain and was purified by two-step ion-exchange chromatography. Using hanging-drop vapour-diffusion crystallization techniques, three different crystal forms of the Fab fragment were obtained in the presence and absence of the transition-state analogue. All crystals diffract X-rays to between 3.5 and 3.2,Å resolution. The two crystal forms grown in the presence of the transition-state analogue contain up to four or eight copies of the Fab in the asymmetric unit and diffract to 3.5 and 3.2,Å, respectively. The crystal of the Fab alone is most likely to contain only two copies of the Fab in the asymmetric unit and diffracts to beyond 3.5,Å. Determination of the structure will provide insights into the active-site arrangement of this antibody and will help to increase our understanding of the molecular mechanisms by which the immune system can evolve catalytic function. [source] A novel noncovalent complex of chorismate mutase and DAHP synthase from Mycobacterium tuberculosis: protein purification, crystallization and X-ray diffraction analysisACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 10 2009Mats Ökvist Chorismate mutase catalyzes a key step in the shikimate-biosynthetic pathway and hence is an essential enzyme in bacteria, plants and fungi. Mycobacterium tuberculosis contains two chorismate mutases, a secreted and an intracellular one, the latter of which (MtCM; Rv0948c; 90 amino-acid residues; 10,kDa) is the subject of this work. Here are reported the gene expression, purification and crystallization of MtCM alone and of its complex with another shikimate-pathway enzyme, DAHP synthase (MtDS; Rv2178c; 472 amino-acid residues; 52,kDa), which has been shown to enhance the catalytic efficiency of MtCM. The MtCM,MtDS complex represents the first noncovalent enzyme complex from the common shikimate pathway to be structurally characterized. Soaking experiments with a transition-state analogue are also reported. The crystals of MtCM and the MtCM,MtDS complex diffracted to 1.6 and 2.1,Å resolution, respectively. [source] Adenosine binding to low-molecular-weight purine nucleoside phosphorylase: the structural basis for recognition based on its complex with the enzyme from Schistosoma mansoniACTA CRYSTALLOGRAPHICA SECTION D, Issue 1 2010Humberto M. Pereira Schistosomes are unable to synthesize purines de novo and depend exclusively on the salvage pathway for their purine requirements. It has been suggested that blockage of this pathway could lead to parasite death. The enzyme purine nucleoside phosphorylase (PNP) is one of its key components and molecules designed to inhibit the low-molecular-weight (LMW) PNPs, which include both the human and schistosome enzymes, are typically analogues of the natural substrates inosine and guanosine. Here, it is shown that adenosine both binds to Schistosoma mansoni PNP and behaves as a weak micromolar inhibitor of inosine phosphorolysis. Furthermore, the first crystal structures of complexes of an LMW PNP with adenosine and adenine are reported, together with those with inosine and hypoxanthine. These are used to propose a structural explanation for the selective binding of adenosine to some LMW PNPs but not to others. The results indicate that transition-state analogues based on adenosine or other 6-amino nucleosides should not be discounted as potential starting points for alternative inhibitors. [source] An Inverse Substrate Orientation for the Regioselective Acylation of 3,,5,-Diaminonucleosides Catalyzed by Candida antarctica lipase B?CHEMBIOCHEM, Issue 8 2005Iván Lavandera Dr. Abstract Candida antarctica lipase B (CAL-B) catalyzes the regioselective acylation of natural thymidine with oxime esters and also the regioselective acylation of an analogue, 3,,5,-diamino-3,,5,-dideoxythymidine with nonactivated esters. In both cases, acylation favors the less hindered 5,-position over the 3,-position by upto 80-fold. Computer modeling of phosphonate transition-state analogues for the acylation of thymidine suggests that CAL-B favors acylation of the 5,-position because this orientation allows the thymine ring to bind in a hydrophobic pocket and forms stronger key hydrogen bonds than acylation of the 3,-position. On the other hand, computer modeling of phosphonamidate analogues of the transition states for acylation of either the 3,- or 5,-amino groups in 3,,5,-diamino-3,,5,-dideoxythymidine shows similar orientations and hydrogen bonds and, thus, does not explain the high regioselectivity. However, computer modeling of inverse structures, in which the acyl chain binds in the nucleophile pocket and vice versa, does rationalize the observed regioselectivity. The inverse structures fit the 5,-, but not the 3,-intermediate thymine ring, into the hydrophobic pocket, and form a weak new hydrogen bond between the O-2 carbonyl atom of the thymine and the nucleophile amine only for the 5,-intermediate. A water molecule might transfer a proton from the ammonium group to the active-site histidine. As a test of this inverse orientation, we compared the acylation of thymidine and 3,,5,-diamino-3,,5,-dideoxythymidine with butyryl acyl donors and with isosteric methoxyacetyl acyl donors. Both acyl donors reacted at equal rates with thymidine, but the methoxyacetyl acyl donor reacted four times faster than the butyryl acyl donor with 3,,5,-diamino-3,,5,-dideoxythymidine. This faster rate is consistent with an inverse orientation for 3,,5,-diamino-3,,5,-dideoxythymidine, in which the ether oxygen atom of the methoxyacetyl group can form a similar hydrogen bond to the nucleophilic amine. This combination of modeling and experiments suggests that such lipase-catalyzed reactions of apparently close substrate analogues like alcohols and amines might follow different pathways. [source] | ||||||||||