Helmut Dietrich

Active site-specific reconstituted copper(II) horse liver alcohol dehydrogenase: a biological model for type 1 Cu2+ and its changes upon ligand binding and conformational transitions.

Insertion of Cu2+ ions into horse liver alcohol dehydrogenase depleted of its catalytic Zn2+ ions creates an artificial blue copper center similar to that of plastocyanin and similar copper proteins. The esr spectrum of a frozen solution and the optical spectra at 296 and 77 K are reported, together with the corresponding data for binary and ternary complexes with NAD+ and pyrazole. The binary complex of the cupric enzyme with pyrazole establishes a novel type of copper proteins having the optical characteristics of Type 1 and the esr parameters of Type 2 Cu2+. Ternary complex formation with NAD+ converts the Cu2+ ion to a Type 1 center. By an intramolecular redox reaction the cuprous enzyme is formed from the cupric enzyme. Whereas the activity of the cupric alcohol dehydrogenase is difficult to assess (0.5%-1% that of the native enzyme), the cuprous enzyme is distinctly active (8% of the native enzyme). The implications of these findings are discussed in view of the coordination of the metal in native copper proteins.

Active site-specifically reconstituted nickel(II) horse liver alcohol dehydrogenase: optical spectra of binary and ternary complexes with coenzymes, coenzyme analogues, substrates, and inhibitors.

Insertion of nickel ions into the empty catalytic site of horse liver alcohol dehydrogenase yields an active enzyme with 65% metal substitution and about 12% intrinsic activity. The electronic absorption spectrum is characterized by bands at 357 nm (2900 M-1 cm-1), 407 nm (3500 M-1 cm-1), 505 nm (300 M-1 cm-1), 570 nm (approximately equal to 130 M-1 cm-1), and 680 nm (approximately equal to 80 M-1 cm-1). The absorption and CD spectra are similar to those of nickel(II) azurin and nickel(II) aspartate transcarbamoylase and prove coordination of the nickel(II) ions to sulfur in a distorted tetrahedral coordination geometry. Changes of the spectra upon ligand binding at the metal or conformation changes of the protein induced by coenzyme, or both, indicate alterations of the metal geometry. The chromophoric substrate trans-4-(N, N-dimethylamino)-cinnamaldehyde forms a ternary complex with Ni(II) liver alcohol dehydrogenase and the coenzyme analogue 1,4,5,6-tetrahydronicotinamide-adenine-dinucleotide, stable between pH 6 and 10. The corresponding ternary complex with NADH is only stable at pH greater than 9.0. The spectral redshifts induced in the substrate are 11 nm larger than those found in the zinc enzyme. We suggest direct coordination of the substrate to the catalytic metal on which acts as a Lewis acid in both substrate coordination and catalysis.

Coordination chemistry and function of the catalytic metal ion in liver alcohol dehydrogenase

A physicochemical characterization of the metal binding center of the active site of the dimeric enzyme horse liver alcohol dehydrogenase (HLADH) has been performed by replacing the zinc ion by various metal ions, e.g. Co, Ni, Cd, Cu. These metal ions have served as spectroscopic and kinetic probes to study the binding of coenzymes, substrates and inhibitors. The metal replacement is performed in two steps. First, the catalytic zinc ions are removed by treatment of crystal suspensions with chelating agents resulting in a species H4Zn(n)2-HLADH (where n denotes the non-catalytic zinc ions which remain in situ). Secondly, the H4Zn(n)2-HLADH can be reconstituted with different metal ions in a crystal suspension or in solution to yield Me(c)2Zn(n)2-HLADH (where c denotes the catalytic metal ion). X-ray crystallographic investigations have shown: (i) in H4Zn(n)2-HLADH only the catalytic zinc ion has been removed without any gross changes in the tertiary structure of HLADH or the ligand sphere of the catalytic metal ion; (ii) on binding NADH to H4Zn(n)2-HLADH, the transition from an ‘open’ to a ‘closed’ conformation occurs analogous to the native enzyme; and (iii) in Co(c)2Zn(n)2-HLADH the cobalt ion has been inserted into the active site accepting a coordination geometry very similar to the zinc ion in the native enzyme. The kinetics and the mechanism of the reconstitution of H4Zn(n)2-HLADH with Co(II), Zn(II) and Ni(II) has been shown to be a two-step process: The spectroscopic characterization of binary and ternary complexes of Me(c)2Zn(n)2-HLADH with coenzymes and/or inhibitors (substrates) has demonstrated that changes of the electronic structure of the catalytic metal ion during enzymatic turn-over are of fundamental importance. A novel kind of interaction of the catalytic metal ion with the coenzyme was noted: the rate of coenzyme dissociation and, therefore, the enzymatic turn-over depends on the kind of metal present in the active site. Concerning the interaction of the catalytic metal ion with non-protein ligands, we conclude: (a) previous conclusions drawn from measurements of the proton relaxation enhancement of the cobalt-bound ligand protons are invalid, since the cobalt ion is not a suitable relaxation probe in this protein; (b) the metal ion acts as a Lewis acid on inner-sphere bound aldehydes; and (c) the coordination number of the catalytic metal ion in binary and ternary complexes of LADH is a matter of controversy. Finally, we point out that previous postulated mechanisms for LADH are premature since the assignment of ionizing groups controlling catalysis is not firmly established. In particular, we have shown by 1H NMR spectroscopy of Co(c)2Zn(n)2-HLADH that the δ(NH) value of the metal ligand His-67 controls the acid-base equilibrium with pKa = 9.2 in the free enzyme. This possibility has never been considered in any previous mechanism.

Spectral evidence for three metal-linked ionization equilibria in the interaction of cobalt(II) horse liver alcohol dehydrogenase with coenzyme and substrate

The visible absorption bands in the region 525-575 nm of the catalytic cobalt ion in cobalt(II) horse liver alcohol dehydrogenase show characteristic pH-dependent changes both in the free enzyme and its complexes with nicotinamide adenine dinucleotide (NAD+) and NAD+ plus ethanol or 2,2,2-trifluoroethanol. In the free enzyme, the change of the coordination environment has an apparent pK of about 9.4. In the binary complex with NAD+ the spectral changes are complex, indicating changes in the coordination sphere in a lower pH range with an estimated pK value of about 7.9. The ternary complexes enzyme X NAD+ X ethanol and enzyme X NAD+ X 2,2,2-trifluoroethanol exhibit very similar, characteristic spectral features; their apparent pK values are 6.3 and less than 4, respectively. We ascribe these pK values to the ionization of the alcohol bound in the ternary complexes. The results demonstrate that the catalytic cobalt ion is sensing changes of the ionization state of the protein when going from low pH forms to high pH forms both in the absence and presence of coenzyme and substrate/inhibitor.

[49] Spectroscopic methods for characterization of immobilized alcohol dehydrogenase

Publisher Summary This chapter provides an account of different spectroscopic methods for the characterization of immobilized horse liver alcohol dehydrogenase (LADH). Absorption spectrophotometry is a suitable and rapid method for the protein determination and for the characterization of immobilized proteins. Fluorometry is another method most frequently used to obtain information on conformational changes in immobilized proteins. Both the spectrophotometric and fluorometric data indicate the far-reaching structural similarity of soluble and Sepharose-bound LADH. The high stability and the identical spectroscopical properties of ternary complexes formed with soluble or immobilized enzyme prove high accessibility of the coenzyme and substrate binding sites and allow a quick determination of their active site contents. Although all these methods cannot prove the degree of homogeneity of the entire population of enzyme molecules, they certainly answer questions about the preservation of active sites, which will be a prerequisite for the further kinetic investigations and practical applications of immobilized enzyme preparations.