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

Abstract

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...

Purification, Substrate Range, and Metal Center of AtzC: the N -Isopropylammelide Aminohydrolase Involved in Bacterial Atrazine Metabolism

“…Since Co(II) has a noninteger spin and is therefore paramagnetic, EPR was a natural choice for studying the metal environment. The EPR (23,26). The observed effects of the inhibitor on the EPR spectra are consistent with Co(II) being present either in the active site or at a distance from the active site where it is influenced by gross protein conformational changes upon inhibitor binding.…”

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

“…Yet, all experimental evidence (crystallographic and spectroscopic) agrees on that the free enzyme in the open conformation is four-coordinate [2][3]10,16,[19][20][21][22]44 . Further, it is widely assumed that the dehydration of the active site that occurs when the enzyme closes is important for the catalytic mechanism 1,2 .…”

“…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.…”

Substrate Specificities of Deuterolysin fromAspergillus oryzaeand Electron Paramagnetic Resonance Measurement of Cobalt-substituted Deuterolysin

“…The g values obtained for Co-deuterolysin were similar to those reported for four-,ˆve-, and six-coordinated cobalt complexes including model and protein systems; some examples are the 4,5,6 compounds. [20][21][22] These complexes, including Co-deuterolysin, have mean g principal values very close to 4, higher than the freeelectron g (2.0023) and close to that of an S＝3 W 2 system. 14) Hyperˆne interaction of the cobalt nucleus also is an important EPR parameter of cobalt complexes.…”