Solvent isotope and viscosity effects on the steady‐state kinetics of the flavoprotein nitroalkane oxidase

Gadda, Giovanni; Fitzpatrick, Paul F.

doi:10.1016/j.febslet.2013.04.021

Cited by 16 publications

(19 citation statements)

References 32 publications

(44 reference statements)

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“…The effect of solvent viscosity on the k cat of wild-type ssIGPS was determined to verify the kinetic mechanisms obtained at different temperatures. In principle, diffusion-limited events such as substrate binding or product dissociation are expected to be sensitive to solvent viscosity while unimolecular chemical transformation steps should impart no viscosity dependence. − Solvent viscosity effects, as defined by the slope of a plot of relative rate versus relative viscosity, were determined for ssIGPS at 25 and 60 °C using glycerol as the viscosogen (Figure ).…”

Section: Resultssupporting

confidence: 92%

Relationship of Catalysis and Active Site Loop Dynamics in the (βα)₈-Barrel Enzyme Indole-3-glycerol Phosphate Synthase

et al. 2018

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It is important to understand how the catalytic activity of enzymes is related to their conformational flexibility. We have studied this activity-flexibility correlation using the example of indole-3-glycerol phosphate synthase from Sulfolobus solfataricus (ssIGPS), which catalyzes the fifth step in the biosynthesis of tryptophan. ssIGPS is a thermostable representative of enzymes with the frequently encountered and catalytically versatile (βα)-barrel fold. Four variants of ssIGPS with increased catalytic turnover numbers were analyzed by transient kinetics at 25 °C, and wild-type ssIGPS was likewise analyzed both at 25 °C and at 60 °C. Global fitting with a minimal three-step model provided the individual rate constants for substrate binding, chemical transformation, and product release. The results showed that in both cases, namely, the application of activating mutations and temperature increase, the net increase in the catalytic turnover number is afforded by acceleration of the product release rate relative to the chemical transformation steps. Measurements of the solvent viscosity effect at 25 °C versus 60 °C confirmed this change in the rate-determining step with temperature, which is in accordance with a kink in the Arrhenius diagram of ssIGPS at ∼40 °C. When rotational diffusion rates of electron paramagnetic spin-labels attached to active site loop β1α1 are plotted in the form of an Arrhenius diagram, kinks are observed at the same temperature. These findings, together with molecular dynamics simulations, demonstrate that a different degree of loop mobility correlates with different rate-limiting steps in the catalytic mechanism of ssIGPS.

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Section: Resultssupporting

confidence: 92%

Relationship of Catalysis and Active Site Loop Dynamics in the (βα)₈-Barrel Enzyme Indole-3-glycerol Phosphate Synthase

et al. 2018

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“…A positive slope of 1.0 is expected if catalysis is fully diffusion-limited by nonchemical steps in catalysis. Alternatively, the horizontal line represents the expected result in the absence of any diffusional limitation [31]. The absence of any appreciable influence of solvent viscosity on k cat or k cat / K M for either substrate ( 3mpa or cys ) clearly demonstrates that Av MDO catalysis is not limited by either substrate binding or product release.…”

Section: Resultsmentioning

confidence: 99%

Non-chemical proton-dependent steps prior to O2-activation limit Azotobacter vinelandii 3-mercaptopropionic acid dioxygenase (MDO) catalysis

Crowell

Sardar

Hossain

et al. 2016

Archives of Biochemistry and Biophysics

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3-mercaptopropionate dioxygenase from Azotobacter vinelandii (Av MDO) is a non-heme mononuclear iron enzyme that catalyzes the O2-dependent oxidation of 3-mercaptopropionate (3mpa) to produce 3-sulfinopropionic acid (3spa). With one exception, the active site residues of MDO are identical to bacterial cysteine dioxygenase (CDO). Specifically, the CDO Arg-residue (R50) is replaced by Gln (Q67) in MDO. Despite this minor active site perturbation, substrate-specificity of Av MDO is more relaxed as compared to CDO. In order to investigate the relative timing of chemical and non-chemical events in Av MDO catalysis, the pH/D-dependence of steady-state kinetic parameters (kcat and kcat/KM) and viscosity effects are measured using two different substrates [3mpa and L-cysteine (cys)]. The pL-dependent activity of Av MDO in these reactions can be rationalized assuming a diprotic enzyme model in which three ionic forms of the enzyme are present [cationic, E(z+1); neutral, Ez; and anionic, E(z−1)]. The activities observed for each substrate appear to be dominated by electrostatic interactions within the enzymatic active site. Given the similarity between MDO and the more extensively characterized mammalian CDO, a tentative model for the role of the conserved ‘catalytic triad’ is proposed.

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“…Error bars represent S.D. Note that the left axis is displayed as a log 10 scale according to convention(40).…”

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confidence: 99%

Evidence for distinct rate-limiting steps in the cleavage of alkenes by carotenoid cleavage dioxygenases

Khadka

Farquhar

Hill

et al. 2019

Journal of Biological Chemistry

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Edited by F. Peter Guengerich Carotenoid cleavage dioxygenases (CCDs) use a nonheme Fe(II) cofactor to split alkene bonds of carotenoid and stilbenoid substrates. The iron centers of CCDs are typically five-coordinate in their resting states, with solvent occupying an exchangeable site. The involvement of this iron-bound solvent in CCD catalysis has not been experimentally addressed, but computational studies suggest two possible roles. 1) Solvent dissociation provides a coordination site for O 2 , or 2) solvent remains bound to iron but changes its equilibrium position to allow O 2 binding and potentially acts as a proton source. To test these predictions, we investigated isotope effects (H 2 O versus D 2 O) on two stilbenoidcleaving CCDs, Novosphingobium aromaticivorans oxygenase 2 (NOV2) and Neurospora crassa carotenoid oxygenase 1 (CAO1), using piceatannol as a substrate. NOV2 exhibited an inverse isotope effect (k H /k D ϳ 0.6) in an air-saturated buffer, suggesting that solvent dissociates from iron during the catalytic cycle. By contrast, CAO1 displayed a normal isotope effect (k H /k D ϳ 1.7), suggesting proton transfer in the rate-limiting step. X-ray absorption spectroscopy on NOV2 and CAO1 indicated that the protonation states of the iron ligands are unchanged within pH 6.5-8.5 and that the Fe(II)-aquo bond is minimally altered by substrate binding. We pinpointed the origin of the differential kinetic behaviors of NOV2 and CAO1 to a single amino acid difference near the solvent-binding site of iron, and X-ray crystallography revealed that the substitution alters binding of diffusible ligands to the iron center. We conclude that solvent-iron dissociation and proton transfer are both associated with the CCD catalytic mechanism. Carotenoid cleavage dioxygenases (CCDs) 2 constitute a family of nonheme iron enzymes that catalyze the cleavage of alk-This work was supported by National Institutes of Health Grants R01EY009339 (to P. D. K.) and R01EY020551 (to J. v. L.), Department of Veterans Affairs Grant IK2BX002683 (to P. D. K.), and Burroughs Wellcome Fund Award 1015187 (to P. D. K.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health or the Department of Veterans Affairs. This article contains Figs. S1-S8 and Tables S1 and S2.

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Solvent isotope and viscosity effects on the steady‐state kinetics of the flavoprotein nitroalkane oxidase

Cited by 16 publications

References 32 publications

Relationship of Catalysis and Active Site Loop Dynamics in the (βα)₈-Barrel Enzyme Indole-3-glycerol Phosphate Synthase

Relationship of Catalysis and Active Site Loop Dynamics in the (βα)₈-Barrel Enzyme Indole-3-glycerol Phosphate Synthase

Non-chemical proton-dependent steps prior to O2-activation limit Azotobacter vinelandii 3-mercaptopropionic acid dioxygenase (MDO) catalysis

Evidence for distinct rate-limiting steps in the cleavage of alkenes by carotenoid cleavage dioxygenases

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Solvent isotope and viscosity effects on the steady‐state kinetics of the flavoprotein nitroalkane oxidase

Cited by 16 publications

References 32 publications

Relationship of Catalysis and Active Site Loop Dynamics in the (βα)8-Barrel Enzyme Indole-3-glycerol Phosphate Synthase

Relationship of Catalysis and Active Site Loop Dynamics in the (βα)8-Barrel Enzyme Indole-3-glycerol Phosphate Synthase

Non-chemical proton-dependent steps prior to O2-activation limit Azotobacter vinelandii 3-mercaptopropionic acid dioxygenase (MDO) catalysis

Evidence for distinct rate-limiting steps in the cleavage of alkenes by carotenoid cleavage dioxygenases

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Relationship of Catalysis and Active Site Loop Dynamics in the (βα)₈-Barrel Enzyme Indole-3-glycerol Phosphate Synthase

Relationship of Catalysis and Active Site Loop Dynamics in the (βα)₈-Barrel Enzyme Indole-3-glycerol Phosphate Synthase