Tunneling of intermediates in enzyme-catalyzed reactions

Weeks, Amanda L.; Lund, L. Koren; Raushel, Frank M.

doi:10.1016/j.cbpa.2006.08.008

Cited by 59 publications

(53 citation statements)

References 33 publications

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“…The conserved features are conformational flexibility, which is a feature not readily identified through sequence or structure alignments, and hydrophobicity, which can be satisfied by many constellations of hydrophobic amino acids. The multiplicity of solutions amongst oxidoreductases is similar to the structural diversity seen for ammonia transfer tunnels in different carbamoylphosphate synthetases (49) and oxygen tunnels in lipoxygenases (15,16).…”

Section: Discussionmentioning

confidence: 55%

The Binding and Release of Oxygen and Hydrogen Peroxide Are Directed by a Hydrophobic Tunnel in Cholesterol Oxidase

et al. 2008

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The usage by enzymes of specific binding pathways for gaseous substrates or products is debated. The crystal structure of the redox enzyme cholesterol oxidase, solved at sub-Ångstrom resolution, revealed a hydrophobic tunnel that may serve as a binding pathway for oxygen and hydrogen peroxide. This tunnel is formed by a cascade of conformational rearrangements and connects the active site with the exterior surface of the protein. To understand the relationship between this tunnel and gas binding and release, three mutant enzymes were constructed to block the tunnel or its putative gate. Mutation of the proposed gating residue Asn485 to Asp or the tunnel residues Phe359 or Gly347 to Trp or Asn, reduces the catalytic efficiency of oxidation. The K mO2 increases from 300 ± 35 μM for the wild-type enzyme to 617 ± 15 μM for the F359W mutant. The k cat for the F359W mutant catalyzed reaction decreases 13-fold relative to the wild-type catalyzed reaction. The N485D and G347N mutants could not be saturated with oxygen. Hydride transfer from the sterol to the flavin prosthetic group is no longer rate limiting for these tunnel mutants. The steady-state kinetics of both wild-type and tunnel-mutant enzymes are consistent with formation of a ternary complex of steroid and oxygen during catalysis. Furthermore, kinetic cooperativity with respect to molecular oxygen is observed with the tunnel mutants, but not with the wild-type enzyme. A rate-limiting conformational change for binding and release of oxygen and hydrogen peroxide, respectively are consistent with the cooperative kinetics. In the atomic resolution structure of F359W, the indole ring of the tryptophan completely fills the tunnel and is only observed in a single conformation. The size of the indole is proposed to limit conformational rearrangement of residue 359 that leads to tunnel opening in the wild-type enzyme. Overall, these results substantiate the functional importance of the tunnel for substrate binding and product release.

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Section: Discussionmentioning

confidence: 55%

The Binding and Release of Oxygen and Hydrogen Peroxide Are Directed by a Hydrophobic Tunnel in Cholesterol Oxidase

et al. 2008

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“…crystallography ͉ structure/function relationships ͉ substrate tunnel ͉ protein film voltammetry ͉ isotope exchange E nzyme channels for substrates are elongated cavities, or ''tunnels,'' which either connect the active site to the solvent or guide intermediates from one active site to another in multifunctional enzymes (1). As far as redox catalysis is concerned, the best-documented example is certainly the 70-Å-long channel that connects the two active sites involved in CO production and utilization in acetyl-CoA synthase/CO dehydrogenase (ACS-CODH) (2,3).…”

mentioning

confidence: 99%

Experimental approaches to kinetics of gas diffusion in hydrogenase

Leroux

Dementin

Burlat

et al. 2008

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

151

238

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Hydrogenases, which catalyze H2 to H ؉ conversion as part of the bioenergetic metabolism of many microorganisms, are among the metalloenzymes for which a gas-substrate tunnel has been described by using crystallography and molecular dynamics. However, the correlation between protein structure and gas-diffusion kinetics is unexplored. Here, we introduce two quantitative methods for probing the rates of diffusion within hydrogenases. One uses protein film voltammetry to resolve the kinetics of binding and release of the competitive inhibitor CO; the other is based on interpreting the yield in the isotope exchange assay. We study structurally characterized mutants of a NiFe hydrogenase, and we show that two mutations, which significantly narrow the tunnel near the entrance of the catalytic center, decrease the rates of diffusion of CO and H2 toward and from the active site by up to 2 orders of magnitude. This proves the existence of a functional channel, which matches the hydrophobic cavity found in the crystal. However, the changes in diffusion rates do not fully correlate with the obstruction induced by the mutation and deduced from the x-ray structures. Our results demonstrate the necessity of measuring diffusion rates and emphasize the role of side-chain dynamics in determining these.crystallography ͉ structure/function relationships ͉ substrate tunnel ͉ protein film voltammetry ͉ isotope exchange

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“…Channeling of substrates has been suggested in a number of metabolic pathways (12)(13)(14)(15)(16)(17). Examples include purine and pyrimidine synthesis, DNA replication, glycolysis, the tricarboxylic acid cycle, lipid metabolism, and amino acid metabolism.…”

mentioning

confidence: 99%

“…Channeling has been established in only a few systems including tryptophan synthase (18) and carbamoylphosphate synthase (19), imidazoleglycerol-phosphate synthase, (20), and 4-hydroxy-2-ketovalerate aldolase/aldehyde dehydrogenase (21) for which both structural and biochemical data are available. These proteins employ "tunnels" to connect active sites (13,14) where they protect reaction intermediates and steer them to a second active site. Allosteric mechanisms aid communication between active sites and thus ensure that production of a reaction intermediate is balanced with respect to its consumption.…”

mentioning

confidence: 99%

An Internal Reaction Chamber in Dimethylglycine Oxidase Provides Efficient Protection from Exposure to Toxic Formaldehyde

Tralau

Lafite

Levy

et al. 2009

Journal of Biological Chemistry

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We report a synthetic biology approach to demonstrate substrate channeling in an unusual bifunctional flavoprotein dimethylglycine oxidase. The catabolism of dimethylglycine through methyl group oxidation can potentially liberate toxic formaldehyde, a problem common to many amine oxidases and dehydrogenases. Using a novel synthetic in vivo reporter system for cellular formaldehyde, we found that the oxidation of dimethylglycine is coupled to the synthesis of 5,10-methylenetetrahydrofolate through an unusual substrate channeling mechanism. We also showed that uncoupling of the active sites could be achieved by mutagenesis or deletion of the 5,10-methylenetetrahydrofolate synthase site and that this leads to accumulation of intracellular formaldehyde. Channeling occurs by nonbiased diffusion of the labile intermediate through a large solvent cavity connecting both active sites. This central "reaction chamber" is created by a modular protein architecture that appears primitive when compared with the sophisticated design of other paradigm substrate-channeling enzymes. The evolutionary origins of the latter were likely similar to dimethylglycine oxidase. This work demonstrates the utility of synthetic biology approaches to the study of enzyme mechanisms in vivo and points to novel channeling mechanisms that protect the cell milieu from potentially toxic reaction products.

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Tunneling of intermediates in enzyme-catalyzed reactions

Cited by 59 publications

References 33 publications

The Binding and Release of Oxygen and Hydrogen Peroxide Are Directed by a Hydrophobic Tunnel in Cholesterol Oxidase

The Binding and Release of Oxygen and Hydrogen Peroxide Are Directed by a Hydrophobic Tunnel in Cholesterol Oxidase

Experimental approaches to kinetics of gas diffusion in hydrogenase

An Internal Reaction Chamber in Dimethylglycine Oxidase Provides Efficient Protection from Exposure to Toxic Formaldehyde

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