On the importance of orientation in general base catalysis by carboxylate

Gandour, Richard D.

doi:10.1016/0045-2068(81)90020-1

Cited by 193 publications

(122 citation statements)

References 47 publications

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“…The second factor that effects the general base catalysis by the carboxylate is the orientation of the carboxylate with respect to the proton to be abstracted. Examination of structure/mechanism studies of several enzymes that employ carboxylate catalysis, a syn orientation is favored over an anti orientation by a factor of between 10 and 100 (Gandour, 1981). Also, Asp165 of the TIM mutant has an anti orientation which probably is largely responsible for the extremely low catalytic activity compared to the syn carboxylate of Glu165 in the wild type (JosephMcCarthy et al, 1994).…”

Section: Discussionmentioning

confidence: 99%

Crystal Structures of the Wild Type and the Glu376Gly/Thr255Glu Mutant of Human Medium-Chain Acyl-CoA Dehydrogenase: Influence of the Location of the Catalytic Base on Substrate Specificity

et al. 1996

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Crystal structures of the wild type human medium-chain acyl-CoA dehydrogenase (MCADH) and a double mutant in which its active center base-arrangement has been altered to that of long chain acyl-CoA dehydrogenase (LCADH), Glu376Gly/Thr255Glu, have been determined by X-ray crystallography at 2.75 and 2.4 Å resolution, respectively. The catalytic base responsible for the R-proton abstraction from the thioester substrate is Glu376 in MCADH, while that in LCADH is Glu255 (MCADH numbering), located over 100 residues away in its primary amino acid sequence. The structures of the mutant complexed with C8-, C12, and C14-CoA have also been determined. The human enzyme structure is essentially the same as that of the pig enzyme. The structure of the mutant is unchanged upon ligand binding except for the conformations of a few side chains in the active site cavity. The substrate with chain length longer than C12 binds to the enzyme in multiple conformations at its ω-end. Glu255 has two conformations, "active" and "resting" forms, with the latter apparently stabilized by forming a hydrogen bond with Glu99. Both the direction in which Glu255 approaches the C R atom of the substrate and the distance between the Glu255 carboxylate and the C R atom are different from those of Glu376; these factors are responsible for the intrinsic differences in the kinetic properties as well as the substrate specificity. Solvent accessible space at the "midsection" of the active site cavity, where the C R -C bond of the thioester substrate and the isoalloxazine ring of the FAD are located, is larger in the mutant than in the wild type enzyme, implying greater O 2 accessibility in the mutant which might account for the higher oxygen reactivity.Acyl-CoA dehydrogenases are a family of flavoenzymes that catalyze the R, -dehydrogenation of acyl-CoA thioesters to the corresponding trans-enoyl-CoA with transfer of reducing equivalents to electron transfer flavoprotein (Beinert, 1963). In mammalian mitochondria, four straight-chain specific acyl-CoA dehydrogenases [short (SCADH)-, medium (MCADH)-, long (LCADH)-, and very long chain (VLCAD) acyl-CoA dehydrogenase] involved in fatty acid catabolism (Beinert, 1963;Aoyama et al., 1994) and three branched-chain specific acyl-CoA dehydrogenases (isovaleryl-, 2-methylbutyryl-, and glutaryl-CoA-DH) in amino acid metabolism have been described (Tanaka & Indo, 1992;Lenich & Goodman, 1986). Among them, MCADH is the most studied: the three dimensional structure of pig MCADH has been determined with and without various substrates and inhibitors (Kim et al., 1993 and its catalytic residue has been identified (Powell & Thorpe, 1988) and confirmed by site-specific mutagenesis (Bross, et al, 1990) and by the three dimensional structures of enzyme-substrate complex (Kim et al., 1993). All seven acyl-CoA dehydrogenases have been cloned, sequenced, and over-expressed in bacterial systems. Of the 27 known amino acid sequences of acylCoA dehydrogenases from both mammalian and bacterial sources available, the catal...

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Section: Discussionmentioning

confidence: 99%

Crystal Structures of the Wild Type and the Glu376Gly/Thr255Glu Mutant of Human Medium-Chain Acyl-CoA Dehydrogenase: Influence of the Location of the Catalytic Base on Substrate Specificity

et al. 1996

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“…The C563Y substitution introduces a bulky tyrosine residue that is likely to interfere with the hydrogen bonding interaction critical for lowering the pK a of D484 to facilitate protonation of the epoxide group (Fig. 3C) (30). We predict that this substitution will inhibit reaction initiation, which would inactivate cyclization.…”

Section: Atlup1-t729f (Yeast)mentioning

confidence: 99%

A conserved amino acid residue critical for product and substrate specificity in plant triterpene synthases

Salmon

Thimmappa

Minto

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Triterpenes are structurally complex plant natural products with numerous medicinal applications. They are synthesized through an origami-like process that involves cyclization of the linear 30 carbon precursor 2,3-oxidosqualene into different triterpene scaffolds. Here, through a forward genetic screen in planta, we identify a conserved amino acid residue that determines product specificity in triterpene synthases from diverse plant species. Mutation of this residue results in a major change in triterpene cyclization, with production of tetracyclic rather than pentacyclic products. The mutated enzymes also use the more highly oxygenated substrate dioxidosqualene in preference to 2,3-oxidosqualene when expressed in yeast. Our discoveries provide new insights into triterpene cyclization, revealing hidden functional diversity within triterpene synthases. They further open up opportunities to engineer novel oxygenated triterpene scaffolds by manipulating the precursor supply.terpenes | natural products | plant defense | cyclization | mutants

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“…groups is also important, but just how important is a subject of controversy (Koshland, 1972;Wang, 1970;Gandour, 1978Gandour, , 1981. Thus, studies of structure and dynamics on chemical models of enzymatic catalysts have the potential of providing information about geometry and its relationship to catalytic efficiency.…”

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confidence: 99%