Catalytic mechanism of Zn2+-dependent polyol dehydrogenases: kinetic comparison of sheep liver sorbitol dehydrogenase with wild-type and Glu154→Cys forms of yeast xylitol dehydrogenase

Klimacek, Mario; Hellmer, Heidemarie; Nidetzky, Bernd

doi:10.1042/bj20061384

Cited by 12 publications

(13 citation statements)

References 34 publications

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“…Biochemical, biophysical, and computational studies have suggested that as zinc is a strong Lewis acid, the water molecule it coordinates would be an hydroxide ion in the binary complex, as proposed for LADH (25), SDH (11,26), and S. solfataricus GlcDH (24). This hydroxide could then act as a base to transiently trap the proton released during formation of the alkoxide.…”

Section: Implications For Active-site Dynamics In Catalysis By the MDmentioning

confidence: 99%

Active site dynamics in the zinc-dependent medium chain alcohol dehydrogenase superfamily

Baker

Britton

Fisher

et al. 2009

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

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Despite being the subject of intensive investigations, many aspects of the mechanism of the zinc-dependent medium chain alcohol dehydrogenase (MDR) superfamily remain contentious. We have determined the high-resolution structures of a series of binary and ternary complexes of glucose dehydrogenase, an MDR enzyme from Haloferax mediterranei. In stark contrast to the textbook MDR mechanism in which the zinc ion is proposed to remain stationary and attached to a common set of protein ligands, analysis of these structures reveals that in each complex, there are dramatic differences in the nature of the zinc ligation. These changes arise as a direct consequence of linked movements of the zinc ion, a zinc-bound bound water molecule, and the substrate during progression through the reaction. These results provide evidence for the molecular basis of proton traffic during catalysis, a structural explanation for pentacoordinate zinc ion intermediates, a unifying view for the observed patterns of metal ligation in the MDR family, and highlight the importance of dynamic fluctuations at the metal center in changing the electrostatic potential in the active site, thereby influencing the proton traffic and hydride transfer events.MDR family ͉ structure ͉ zinc metalloenzyme ͉ reaction mechanism Z inc-dependent enzymes catalyze many important cellular processes (1); yet, despite this, the role played by the zinc ion in catalysis is not completely understood, partly because zinc is silent in a range of useful spectroscopic techniques, such as EPR and optical spectroscopy. One of the best characterized zinccontaining enzyme families is the Zn-dependent medium chain alcohol dehydrogenase superfamily (MDR), which catalyzes the oxidation of primary or secondary alcohols to the corresponding aldehydes or ketones using NAD(P) ϩ as a cofactor (2).The structures of many MDR family members have been determined with that of liver alcohol dehydrogenase (LADH) as the prototypical member (3). These studies have shown that the subunit of these enzymes is constructed from two domains, a nucleotide binding domain and a catalytic domain (4), with the essential zinc ion located deep in the cleft between them. The ligands to the zinc are provided by residues from the catalytic domain and, in the absence of substrate, the zinc is coordinated to the side chains of three well conserved residues, which in LADH are Cys-46, His-67, and Cys-174 (2). The tetrahedral coordination shell of the zinc is completed by a water molecule (2).In the classical text book mechanism for this enzyme family (5), this zinc-bound water molecule is suggested to be displaced by the incoming hydroxyl group of the alcohol on substrate binding, to leave the zinc ion coordinated to the substrate and the same three protein ligands as in the apo enzyme. The redox reaction requires the net removal of two hydrogen atoms from the substrate and is thought to proceed via proton loss from the substrate hydroxyl to bulk solvent by using a proton relay system to form an alkoxide intermedia...

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Section: Implications For Active-site Dynamics In Catalysis By the MDmentioning

confidence: 99%

Active site dynamics in the zinc-dependent medium chain alcohol dehydrogenase superfamily

Baker

Britton

Fisher

et al. 2009

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

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“…1 In the many cases where evolutionary pressures have equalized the relative barriers to isotope-independent substrate binding and product release steps and to isotope-dependent hydride transfer steps, 2 no single step is rate determining and the intrinsic kinetic isotope effect (KIE) is suppressed. 3 Apparent intrinsic 1°DKIE have been reported for enzymatic hydride transfer at nonphysiological substrates, 4 or for catalysis by enzymes crippled by site-directed mutagenesis. 4b The effect of these perturbations in substrate and enzyme structure on the intrinsic 1°DKIE for the unperturbed reaction is unclear.…”

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confidence: 99%

Structure–Reactivity Effects on Intrinsic Primary Kinetic Isotope Effects for Hydride Transfer Catalyzed by Glycerol-3-phosphate Dehydrogenase

2016

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Primary deuterium kinetic isotope effects (1°DKIE) on (kcat/KGA, M–1 s–1) for dianion (X2–) activated hydride transfer from NADL to glycolaldehyde (GA) catalyzed by glycerol-3-phosphate dehydrogenase were determined over a 2100-fold range of enzyme reactivity: (X2–, 1°DKIE); FPO32–, 2.8 ± 0.1; HPO32–, 2.5 ± 0.1; SO42–, 2.8 ± 0.2; HOPO32–, 2.5 ± 0.1; S2O32–, 2.9 ± 0.1; unactivated; 2.4 ± 0.2. Similar 1°DKIEs were determined for kcat. The observed 1°DKIEs are essentially independent of changes in enzyme reactivity with changing dianion activator. The results are consistent with (i) fast and reversible ligand binding; (ii) the conclusion that the observed 1°DKIEs are equal to the intrinsic 1°DKIE on hydride transfer from NADL to GA; (iii) similar intrinsic 1°DKIEs on GPDH-catalyzed reduction of the substrate pieces and the whole physiological substrate dihydroxyacetone phosphate. The ground-state binding interactions for different X2– are similar, but there are large differences in the transition state interactions for different X2–. The changes in transition state binding interactions are expressed as changes in kcat and are proposed to represent changes in stabilization of the active closed form of GPDH. The 1°DKIEs are much smaller than observed for enzyme-catalyzed hydrogen transfer that occurs mainly by quantum-mechanical tunneling.

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“…Between them there is a deep cleft that hosts the catalytically requisite Zn 2+ ion and the cofactor. These protein ISSN 1399-0047 # 2015 International Union of Crystallography subunits associate into homodimeric assemblies in mammalian ADHs or into homotetramers in fungal, bacterial and yeast ADHs (Eklund & Ramaswamy, 2008) and also in PDHs (Klimacek et al, 2007). Together with the catalytic zinc ion, most dehydrogenases from this superfamily have another zinc which is also located in the catalytic domain, specifically within the so-called lobe loop, which is defined as the structural zinc (Auld & Bergman, 2008) and whose functional role, if any, is not yet clear (Raj et al, 2014).…”

Section: Introductionmentioning

confidence: 99%

“…In this regard, uncertainties remain about the functional roles of the acidic residues Glu60 (using GPDH numbering) and Glu144, both of which are conserved among the PDHs (Eklund et al, 1985;Jeffery & Jö rnvall, 1988) and which are very close to the catalytic zinc. Whereas in this latter family of enzymes Glu60 (Glu68 in horse liver ADH; hlADH) is a ligand of Zn 2+ (Klimacek et al, 2007), Glu144 (Cys174 in hlADH) is linked to the zinc ion through a water molecule (also conserved in the family) and thus belongs to the second coordination sphere. In the case of hlADH, Glu68, which does not coordinate to the metal in the absence of substrates (the ligands of the zinc are Cys46, His67, Cys174 and a water molecule), has been suggested to play different roles, namely a stabilizing role in the active site (Al-Karadaghi et al, 1994), as a modulator of the electrostatic field in this environment (Ganzhorn & Plapp, 1988) and also as a transient ligand of the zinc during the catalytic cycle, permitting efficient product release (Ryde, 1995).…”

Section: Introductionmentioning

confidence: 99%

Enantioselective oxidation of galactitol 1-phosphate by galactitol-1-phosphate 5-dehydrogenase fromEscherichia coli

Benavente

Esteban-Torres

Kohring

et al. 2015

Acta Cryst D Biol Crystallogr

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Galactitol-1-phosphate 5-dehydrogenase (GPDH) is a polyol dehydrogenase that belongs to the medium-chain dehydrogenase/reductase (MDR) superfamily. It catalyses the Zn(2+)- and NAD(+)-dependent stereoselective dehydrogenation of L-galactitol 1-phosphate to D-tagatose 6-phosphate. Here, three crystal structures of GPDH from Escherichia coli are reported: that of the open state of GPDH with Zn(2+) in the catalytic site and those of the closed state in complex with the polyols Tris and glycerol, respectively. The closed state of GPDH reveals no bound cofactor, which is at variance with the conformational transition of the prototypical mammalian liver alcohol dehydrogenase. The main intersubunit-contacting interface within the GPDH homodimer presents a large internal cavity that probably facilitates the relative movement between the subunits. The substrate analogue glycerol bound within the active site partially mimics the catalytically relevant backbone of galactitol 1-phosphate. The glycerol binding mode reveals, for the first time in the polyol dehydrogenases, a pentacoordinated zinc ion in complex with a polyol and also a strong hydrogen bond between the primary hydroxyl group and the conserved Glu144, an interaction originally proposed more than thirty years ago that supports a catalytic role for this acidic residue.

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Catalytic mechanism of Zn2+-dependent polyol dehydrogenases: kinetic comparison of sheep liver sorbitol dehydrogenase with wild-type and Glu154→Cys forms of yeast xylitol dehydrogenase

Cited by 12 publications

References 34 publications

Active site dynamics in the zinc-dependent medium chain alcohol dehydrogenase superfamily

Active site dynamics in the zinc-dependent medium chain alcohol dehydrogenase superfamily

Structure–Reactivity Effects on Intrinsic Primary Kinetic Isotope Effects for Hydride Transfer Catalyzed by Glycerol-3-phosphate Dehydrogenase

Enantioselective oxidation of galactitol 1-phosphate by galactitol-1-phosphate 5-dehydrogenase fromEscherichia coli

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