Solvent deuterium isotope effect on the liver alcohol dehydrogenase reaction

Schmidt, Jack; Chen, Judy; DeTraglia, Michael C.; Minkel, Daniel T.; McFarland, James T.

doi:10.1021/ja00507a033

Cited by 42 publications

(38 citation statements)

References 10 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In a recent reevaluation of the pH and isotope effects on the oxidation of alcohols by LADH; Cook and Cleland (36) This interpretation of kinetic results is compatible with the results ofour spectroscopic studies. However, the pKa controlling alcohol oxidation at the ternary complex level has been assigned to ionization of the metal-bound substrate in a tetracoordinate species (6,7), to the metal-bound water molecule in a pentacoordinate species (8,9), and to histidine-51 (36). Since the important chemical step of hydride transfer in the LADH-catalyzed oxidation of alcohol is separated from the rate-limiting release of products by this ionization, assignment of the group and of the catalytic role of the metal-bound water molecule remains an important problem in understanding the mechanism of action of LADH.…”

Section: Discussionmentioning

confidence: 99%

“…The suggestion has been made on the basis of proton relaxation enhancement studies of enzyme-coenzyme-inhibitor complexes that substrates bind as outer sphere ligands only with no change in coordination number (5). Interpretations of the pH dependence of the kinetic parameters of the enzyme have invoked formation of both tetracoordinate (6, 7) and pentacoordinate (8,9) intermediates as the catalytically active species. Difference Fourier syntheses of (alcohol) inhibitor substrate analogs bound to the active site of the enzyme-NAD' complex crystallized in the presence of 2-methyl-2,4-pentanediol suggest that the substrate displaces the metal-bound water molecule to form a tetracoordinate species only (10).…”

mentioning

confidence: 99%

See 1 more Smart Citation

Coordination environment of the active-site metal ion of liver alcohol dehydrogenase.

Makinen¹,

Yim²

1981

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

View full text Add to dashboard Cite

The coordination environment of the catalytically active metal ion of horse liver alcohol dehydrogenase (alcohol:NAD+ oxidoreductase, EC 1.1.1.1) has been investigated by electron paramagnetic resonance (EPR) methods with use of the active-site-specific Co2+-reconstituted enzyme. The EPR absorption spectrum of the metal-substituted enzyme is characteristic of a rhombically distorted environment. The spectrum of the enzyme-NAD+ complex shows approximate axial symmetry of the metal ion site, indicating that binding of the coenzyme induces a structural alteration in the active-site region. This environment is not significantly altered further by binding of the competitive inhibitor pyrazole. To assign the coordination number of the active-site metal ion, the zero-field splitting was determined on the basis ofthe temperature dependence ofthe spin-lattice relaxation of the Co2+ ion. The zero-field splitting energies are '9 cm-' for the free Co2+-reconstituted enzyme and e46 and -47 cm-' for the enzyme-NAD+ and enzyme-NAD+-pyrazole complexes, respectively. On the basis of studies of structurally defined small molecule complexes, these values are compatible with a tetracoordinate metal ion in the active site of the free enzyme but a pentacoordinate metal ion in the binary enzyme-NAD+ complex and in the ternary enzyme-NAD+-inhibitor complex and, therefore, presumably also in the catalytically active ternary enzyme-NAD+-alcohol complex formed in the course of alcohol oxidation.The oxidation ofalcohol catalyzed by liver alcohol dehydrogenase (LADH; alcohol:NAD' oxidoreductase, EC 1.1.1.1) proceeds at the level of a ternary complex formed by the enzyme, the coenzyme NAD', and the substrate as the catalytically active intermediate of the reaction (1-3). In the free enzyme, the tetracoordinate Zn2+ ion in the active site is ligated by the sulfhydryl groups of cysteine-46 and cysteine-174, the NE atom of histidine-67, and a water molecule (4). The coordination number of the active-site metal ion is considerably less well assigned for reaction intermediates. The suggestion has been made on the basis of proton relaxation enhancement studies of enzyme-coenzyme-inhibitor complexes that substrates bind as outer sphere ligands only with no change in coordination number (5). Interpretations of the pH dependence of the kinetic parameters of the enzyme have invoked formation of both tetracoordinate (6, 7) and pentacoordinate (8, 9) intermediates as the catalytically active species. Difference Fourier syntheses of (alcohol) inhibitor substrate analogs bound to the active site of the enzyme-NAD' complex crystallized in the presence of 2-methyl-2,4-pentanediol suggest that the substrate displaces the metal-bound water molecule to form a tetracoordinate species only (10). However, the crystallizing reagent 2-methyl-2,4-pentanediol acts as substrate analog of the enzyme, resulting in a mixture of enzyme-NAD' and enzyme-NADH species in the crystal.* Under these conditions, addition of alcohol substrates may lead also to formation o...

show abstract

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Coordination environment of the active-site metal ion of liver alcohol dehydrogenase.

Makinen¹,

Yim²

1981

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

View full text Add to dashboard Cite

show abstract

“…This conclusion has strong implications on the reaction mechanism of alcohol dehydrogenase. Several mechanisms have been proposed that involve five-coordinate structures as significant intermediates [6][7][8][9][10][11][12][13] . The present calculations do not support such proposals; since a 46 kJ/mole energy difference corresponds to an equilibrium constant of 1 .…”

Section: The Relative Stability Of Four-and Five-coordinate Zinc Compmentioning

confidence: 99%

“…Several mechanistic proposals have been put forward, however, according to which fivecoordinate intermediates play an essential role during catalysis [6][7][8][9][10][11][12][13] . Crystallographic and spectroscopic data showing that binding to zinc of certain bidentate inhibitors is fivecoordinate 14,15 have been taken as evidence in favor of these suggestions.…”

Section: Introductionmentioning

confidence: 99%

“…Crystallographic and spectroscopic data showing that binding to zinc of certain bidentate inhibitors is fivecoordinate 14,15 have been taken as evidence in favor of these suggestions. Furthermore, spectroscopic studies of metal-substituted alcohol dehydrogenase have indicated that several binary and ternary complexes may be five-coordinate [6][7][8][15][16][17][18][19][20] , although other spectroscopic investigations contradict these results [21][22][23][24][25] . The kinetic evidence is also scattered and has been taken to favor fourcoordination 1,26 , as well as five-coordination 6,7,9,11,27 , of zinc in the catalytically productive ternary complexes.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Molecular dynamics simulations of alcohol dehydrogenase with a four‐ or five‐coordinate catalytic zinc ion

1995

View full text Add to dashboard Cite

A detailed parameterization is presented of a zinc ion with one histidine and two cysteinate ligands, together with one or two water, hydroxide, aldehyde, alcohol, or alkoxide ligands. The parameterization is tailored for the active site of alcohol dehydrogenase and is obtained entirely from quantum chemical computations. The force‐field reproduces excellently the geometry of quantum chemically optimized zinc complexes as well as the crystallographic geometry of the active site of alcohol dehydrogenase and small organic structures. The parameterization is used in molecular dynamics simulations and molecular mechanical energy minimizations of alcohol dehydrogenase with a four‐ or five‐coordinate catalytic zinc ion. The active‐site zinc ion seems to prefer four‐coordination over five‐coordination by at least 36 kJ/mol. The only stable binding site of a fifth ligand at the active‐site zinc ion is opposite to the normal substrate site, in a narrow cavity behind the zinc ion. Only molecules of the size of water or smaller may occupy this site. There are large fluctuations in the geometry of the zinc coordination sphere. A four‐coordinate water molecule alternates frequently (every 7 ps) between the substrate site and the fifth binding site and even two five coordinate water molecules may interchange ligation sites without prior dissociation. Ligand exchange at the zinc ion probably proceeds by a dissociative mechanism. The results show that it is essential to allow for bond stretching degrees of freedom in molecular dynamics simulations to get a correct description of the dynamics of the metal coordination sphere; bond length constraints may restrict the accessible part of the phase space and therefore lead to qualitatively erroneous results. © 1995 Wiley‐Liss, Inc.

show abstract

The Metallobiochemistry of Zinc Enzymes

Vallée

Galdes

1984

Advances in Enzymology - And Related Areas of Molecular Biology

175

View full text Add to dashboard Cite

The honour and opportunity of being invited to address you on the occasion of Professor Houtman's retirement is very much appreciated. However, having met him yesterday, I am convinced that I am not here to help celebrate his retirement as much as to initiate his new career.Just like careers, the role of metals in biology can be viewed from multiple points of view. In the past, metals have variously been thought to be beneficial, toxic or to serve no purpose at all in biology. It is not very long ago that documentation for the validity of all th ree perceptions seemed to coexist, and that they had equally vigorous advocates; but by now the hypothesis that they serve no biological purpose has faded into oblivion. This dilemma owed much to the fact that many metals are present in 'trace' amounts. I want to address myself today to the general principles underlying the evolution of metallobiochemistry and of its subdisciplines which did not exist a generation ago and give perspective to metals as toxic, environmental agents, a subject which has been of special interest to Prof. Houtman.The word 'trace' implies something that can be measured qualitatively but not quantitatively, and until about a generation ago that was true for many metals in biology. Therefore it was a nearly mystical subject explored of ten by individuals who had ideas about the problem but who performed few experiments to verify or reject them. This has changed. Measurement of very low concentrations of metals in biology has become quite routine. Technical problems have been eliminated to the point where the term 'trace' has become meaningless. That is fortunate, indeed, because to many 'trace' also meant 'unimportant' or 'insignificant' .Attention to the detection limits of analytical methods for metals has become almost irrelevant now. Yet, the fact that the II-B metals and those of the first transition series all occur in biological matter in similarly low concentrations led to the tacit inference that all of them would be found to perform similar functions. Such an emphasis on similarities of concentrations completely ignored the chemical properties of metals which are unique for each. Not too surprisingly, their biological roles have turned out to be as characteristic as their chemistries, enhanced by co-ordination of metals with 132 12 (1988) DELFT PROGRESS REPORT

show abstract

Solvent deuterium isotope effect on the liver alcohol dehydrogenase reaction

Cited by 42 publications

References 10 publications

Coordination environment of the active-site metal ion of liver alcohol dehydrogenase.

Coordination environment of the active-site metal ion of liver alcohol dehydrogenase.

Molecular dynamics simulations of alcohol dehydrogenase with a four‐ or five‐coordinate catalytic zinc ion

The Metallobiochemistry of Zinc Enzymes

Contact Info

Product

Resources

About