Crystal structure determinations of coenzyme analog and substrate complexes of liver alcohol dehydrogenase: binding of 1,4,5,6-tetrahydronicotinamide adenine dinucleotide and trans-4-(N,N-dimethylamino)cinnamaldehyde to the enzyme

Cedergren-Zeppezauer, Eila; Samama, Jean‐Pierre; Eklund, Hans

doi:10.1021/bi00263a011

Cited by 99 publications

(94 citation statements)

References 20 publications

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“…The low rate of catalytic hydride transfer from NADH to DACA can be related to electronic properties of the substrate which are of little significance for its binding, however, and does not reflect substrate binding in an atypical (low-productive) mode. On the contrary, it has been well established that DACA is bound at the same site and through similar bonding interactions as aldehydes in general [2,22]. If the coenzyme association process is assumed to be generally affected by aldehyde binding at this site, then such effects should be particularly pronounced with DACA which interacts exceptionally strongly with the site.…”

Section: General Characteristics Of Nadh and Aldehyde Bindingmentioning

confidence: 99%

“…3 and 5, is associated with a mechanistically important change in the binding interactions between enzyme and DACA. The catalytically crucial inner-sphere coordination of DACA to the active-site zinc ion [16, 22,23] is not detectably at hand in the binary enzyme. DACA complex, but requires the presence of bound coenzyme.…”

Section: Synergism Between Nadh and Daca Bindingmentioning

confidence: 99%

“…aldehyde pathway for ternary-complex formation is fully kinetically competent in the sense that it is not impaired or blocked through low rates of substrate and/or coenzyme association. When the reaction proceeds by this pathway, the coenzyme association step must include concomitant ligand exchange at the catalytic zinc ion (such that DACA is transferred from the outer to the inner coordination sphere of the metal with displacement of zinc-bound water [16,22]). Results in Fig.…”

Section: Synergism Between Nadh and Daca Bindingmentioning

confidence: 99%

“…DACA complex (irrespective of what pathway is utilized) cannot be rate limited by any first-order process slower than 1000 s-'. Conformational changes induced by NADH binding or otherwise associated with ternary-complex formation [22,23], as well as ligand exchangc at the catalytic zinc ion, must show rate constants exceeding this value. This confirms our previous conclusion [24] that the observed limitation at about 250 s p l of the rate constant for complex formation between enzyme and zinc-chelating reagents cannot be attributed to a slow step of water dissociation from the catalytic zinc ion [25,26].…”

Section: Synergism Between Nadh and Daca Bindingmentioning

confidence: 99%

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Catalytic significance of binary enzyme‐aldehyde complexes in the liver alcohol dehydrogenase reaction

Andersson

Kvassman

Oldén

et al. 1984

European Journal of Biochemistry

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1. The interaction of liver alcohol dehydrogenase with NADH and aldehyde substrates has been characterized with respect to ternary-complex formation by the apparently non-preferred pathway which involves intermediate formation of binary enzyme . aldehyde complexes. Rate constant estimates are reported for dimethylaminocinnamaldehyde (DACA) binding to free enzyme and for NADH binding to the enzyme . DACA complex.2. The rate ofNADH (or NAD') association to liver alcohol dehydrogenaseis not detectably affected by DACA binding to the enzyme, but the NADH dissociation rate decreases approximately by a factor of 6. The NADHinduced increase in affinity of the enzyme for DACA is similarly attributable to a decreased dissociation rate rather than an increased association rate of the aldehyde. DACA dissociates much more rapidly than coenzyme from the enzyme. NADH . aldehyde complex and shows a higher association rate constant than NADH in its interaction with free enzyme.3. It is concluded from these results that the enzymic reduction of typical aldehyde substrates will conform to a rate equation which is experimentally indistinguishable from that of a compulsory-order mechanism with coenzyme binding preceding substrate binding, and that this rate equation will obtain irrespective of which pathway for ternary-complex formation is actually preferred. Rate equations provide no reliable information about the order of ligand binding in ternary-complex systems.4. A flow analysis is presented which indicates that coenzyme and substrate are actually bound in random order to liver alcohol dehydrogenase during the enzymic reduction of aldehydes by NADH. The enzyme. aldehyde pathway for ternary-complex formation is fully kinetically competent, and reaction flow via this pathway may predominate when aldehyde concentrations exceed those required for half-saturation of free enzyme. Binary enzyme . aldehyde complexes are seemingly insignificant with respect to the rate behaviour of the enzyme, but may provide most significant and even predominant contributions to the catalytic reaction flow.Liver alcohol dehydrogenase [I ] catalyzes alcohol/aldehyde interconversion by a ternary-complex mechanism with NAD+/NADH as coenzymes. The enzyme shows a significant affinity for aldehyde substrates whether or not NADH is present (and vice versa) [1,2], which indicates that the productive enzyme. NADH . aldehyde complexes can be assumed to be formed by a basically random-order binding mechanism (Scheme 1). The enzyme. aldehyde pathway for ternary-complex formation in Scheme 1 does not appear to contribute appreciably to the catalytic reaction flow, however. The enzymic reduction of aldehydes by NADH invariably has been found to conform to the rate equation of a compulsoryorder mechanism with coenzyme binding preceding binding of the aldehyde substrate [3 -61.The reason why this binding order appears to be preferred remains obscure. Essentially no information is available about the effect of coenzyme on the kinetics of substrate binding, or about th...

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Section: General Characteristics Of Nadh and Aldehyde Bindingmentioning

confidence: 99%

Section: Synergism Between Nadh and Daca Bindingmentioning

confidence: 99%

Section: Synergism Between Nadh and Daca Bindingmentioning

confidence: 99%

Section: Synergism Between Nadh and Daca Bindingmentioning

confidence: 99%

See 2 more Smart Citations

Catalytic significance of binary enzyme‐aldehyde complexes in the liver alcohol dehydrogenase reaction

Andersson

Kvassman

Oldén

et al. 1984

European Journal of Biochemistry

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“…Many kinetic data and results from X-ray crystallographic studies on binary and ternary complexes are available for HLADH [6][7][8][9][10][11][12][13], making this enzyme one of the best characterized dehydrogenases. Several groups have performed modelling studies on this enzyme system, trying to obtain more details about the catalytic and structural features of HLADH [14][15][16].…”

Section: Introductionmentioning

confidence: 99%

Kinetic and modelling studies of NAD+ and poly(ethylene glycol)-bound NAD+ in horse liver alcohol dehydrogenase

Vanhommerig

Sluyterman

Meijer

1996

Biochimica et Biophysica Acta (BBA) - Protein Structure and Mol

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Zn‐Dependent Medium‐Chain Dehydrogenases/Reductases

Meijers

Cedergren-Zeppezauer

2004

Handbook of Metalloproteins

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Medium‐chain dehydrogenases/reductases (MDRs) have about 350 to 390 amino acids and belong to a large superfamily of proteins including the Zn‐dependent alcohol dehydrogenases (ADHs) and several other Zn‐dependent activities. They are dimers or tetramers and utilize either NADH or NADPH as electron carriers referred to as cofactors. The oxidation/reduction process is a 2‐electron/1‐proton (hydride ion, H − ) transfer between substrate and cofactor. The compounds oxidized or reduced by these enzymes vary considerably in structure ranging from small primary and secondary alcohols/aldehydes/ketones (aliphatic and aromatic), sugars (sorbitol/fructose, glucose) to large substances like fatty acids, steroids, and retinoids. ADHs are widely distributed in nature and some MDR members are found in all species. An essential Zn ion at the active site is common to all MDRs but some members have two zinc ions per subunit. The 3D Structures of Zn‐dependent MDRs are highly similar and they essentially have the same polypeptide fold. The low sequence identity found sometimes could not immediately reveal this close structural relationship. Now numerous structures have been examined indicating that the evolutionary strategy for MDRs has been to vary amino acids preferably at the reaction center and further away in the substrate binding area. Thus, the size and shape of the substrate channel is adapted for small or large substrates, hydrophilic or hydrophobic substances, modifications attained via few mutations, or significant deletions/additions of amino acids. A further opportunity to create multiplicity has been to vary the control mechanism for the protein conformation change and the oligomeric state. Some enzymes undergo significant structural changes upon cofactor binding, while others do not. A transition state intermediate of NADH in complex with horse liver alcohol dehydrogenase, resolved to atomic resolution (≈1 Å), has revealed structural modifications of the cofactor associated with the activation process for hydride transfer. This involves an OH − pyridine ring adduct, connected to the zinc center. The distortion of the nicotinamide at the enzyme site, affected by the presence of the negatively charged oxygen ligand, influences three parameters: (i) the puckering of the ring, (ii) a deviation from a standard carbon–carbon double bond length, and (iii) accumulation of negative charge at the C4 atom promoting hydride transfer. The active site Zn‐center of this large enzyme group shows great flexibility with respect to metal–ligand geometry and dynamics. Structural and spectroscopic evidence suggest that Zn‐dependent MDRs should be included in the general family of Zn‐enzymes, which require water and hydroxide ion as reactants during catalysis.

show abstract

Crystal structure determinations of coenzyme analog and substrate complexes of liver alcohol dehydrogenase: binding of 1,4,5,6-tetrahydronicotinamide adenine dinucleotide and trans-4-(N,N-dimethylamino)cinnamaldehyde to the enzyme

Cited by 99 publications

References 20 publications

Catalytic significance of binary enzyme‐aldehyde complexes in the liver alcohol dehydrogenase reaction

Catalytic significance of binary enzyme‐aldehyde complexes in the liver alcohol dehydrogenase reaction

Kinetic and modelling studies of NAD+ and poly(ethylene glycol)-bound NAD+ in horse liver alcohol dehydrogenase

Zn‐Dependent Medium‐Chain Dehydrogenases/Reductases

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