Chemical reactivities of catalytic and noncatalytic zinc or cobalt atoms of horse liver alcohol dehydrogenase: differentiation by their thermodynamic and kinetic properties.

Abstract

Horse liver alcohol dehydrogenase (EC 1.1.1.1) contains one catalytic and one noncatalytic pair of zinc atoms that can be replaced selectively with cobalt and/ or 65zinc. We have now prepared "hybrid" metalloenzymes by specifically replacing one or both pairs of zinc atoms with Wzinc and/or cobalt. Their differential chemical reactivities serve to characterize the metal atoms at either site. The spectral and kinetic properties of the resultant 65zinc, cobalt, and hybrid enzymes, as well as those of their complexes with 1,10-phenanthroline, identify the metal atoms that are at the catalytic sites and differentiate them from those at the noncatalytic sites. All data are in complete agreement with the results of the x-ray crystal structure analysis. Remarkably, under the conditions used, chemical reactivity, as gauged by thermodynamic methods under equilibrium conditions, identifies the catalytic metal atoms as those which are reactive to I,10-phenanthroline, while this reagent does not affect the noncatalytic pair. Under dynamic conditions the kinetics of the metal-metal exchange reveals the converse to be true: the chemical reactivity of the noncatalytic atoms is much higher and, hence, they exchange more rapidly. The results are examined in terms of thermodynamic and kinetic properties of metal complex ions which serve as the basis of possible mechanisms underlying these observations. Chemical modifications of particular amino-acid side chains affect the activities of enzymes, linking their chemical to their catalytic properties and differentiating functional from other residues. Such differentiation may be the result of thermodynamic, kinetic, or both these features of the reactants. In this context, the characteristic properties of the metals of metalloenzymes enable their use as spectral, kinetic, and spectrokinetic probes (1). The realization that many enzymes may contain different classes of metal atoms allows an extension of these approaches to the delineation of different chemical reactivities of metals in terms of spectra or rates of metal-metal exchange, the interaction of metals with ligands, the environment of metals, and their modes of interaction with substrates or inhibitors.One such enzyme, horse liver alcohol dehydrogenase, LADH, (EC 1.1.1.1) contains four zinc atoms (2, 3) two of which are "catalytic" and two of which are "noncatalytic" or structural, as judged by kinetic, spectroscopic, and chemical evidence (3-5). On this basis, we have designated these Abbreviations: [(LADH)Zn2Zn2], native horse liver alcohol dehydrogenase; OP, 1,10-phenanthroline. In order to differentiate and clarify presentation, the first pair of metal (Me) atoms in the standard formulation is designated the "N" (noncatalytic) and the second the "C" (catalytic) pair, i.e., l(LADH)N2C2I. Hence two classes of zinc as "C" and "N," respectively, and will refer to them in this manner throughout this report. X-ray crystal structure analysis reveals the details of the ligands binding to these pairs of zinc atoms: those ...

L-histidine inhibits production of lysophosphatidic acid by the tumor-associated cytokine, autotaxin

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Zinc binding to peptide analogs of the structural zinc site in alcohol dehydrogenase: Implications for an entatic state

“…This type of oxidoreductive interplay between thiols/disulfides has been shown to be a general phenomenon suggested to be associated with zinc transport in metallothionein and/or metalloproteins in general [14,15]. Results of early studies removing this zinc by dialysis were believed to establish its structural importance [6,7,16]. Enzymes of related type but not containing this zinc site, e.g., sorbitol dehydrogenase, differ in quaternary structure [17,18], and recombinant enzyme variants of ADH, with one each of the zinc ligands exchanged from Cys to Ala, are labile [19].…”

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