Zinc binding of alcohol and sorbitol dehydrogenases.

Abstract

Zinc is an essential component of many enzymes, serving a role for catalytic activity or structural stability. The removal of catalytic zinc results in an inactive apoenzyme which, however, often retains the native tertiary structure. Structural zinc frequently contributes to the maintenance of the structure of oligomeric enzymes. The removal of zinc from such proteins therefore prevents subunit association. As discerned from zinc analysis of structurally investigated zinc metalloenzymes, the characteristics of a catalytic zinc-binding motif, in many cases, is a combination of three His/Glu/Asp/Cys residues and an activated H2O-molecule (Vallee & Auld, 1990). The spacers between the first and second ligands are short, typically 1–3 amino acids long (alcohol dehydrogenase and sorbitol dehydrogenase are exceptions with 21–25 residues). The second spacer, longer in nature, separates the second and third ligands by about 20–120 amino acid residues (Vallee & Auld, 1989). The structural zinc is necessary for activity only to the extent that the overall conformation of the enzyme affects its action. It serves as a cross-linking agent to stabilize structures (Berg, 1987). The observed pattern of ligands frequently encompasses four cysteine residues, closely spaced in the linear amino acid sequence (Vallee & Auld, 1990). Sorbitol dehydrogenase (SDH) harbours one catalytic zinc atom per subunit (Jeffery et al., 1984a). Because of the structural relationship between SDH and alcohol dehydrogenase (ADH), two of the three ligands to the zinc could be established early in SDH (Cys44 and His69, Jeffery et al., 1984b). The homology in the area around the third zinc ligand in SDH, though, was low toward ADH. The modelled structure of sheep SDH using the crystal structure of horse ADH as reference, gave the best fit with a glutamic acid residue (Glu 155) as the third zinc ligand (Eklund et al., 1985). In order to establish the exact nature of the third zinc ligand, a set of five different potential ligands were mutated to Ala or Gln, residues not able to ligand zinc, and the proteins were expressed in E. coli with subsequent purification and determination of enzyme activities and zinc contents (Karlsson, 1994). ADH contains two zinc atoms per subunit, one catalytic and one structural (Akeson, 1964; Drum et al., 1969; Eklund et al., 1976). The binding site of the structural zinc atom in ADH involves four cysteine residues at positions 97, 100, 103, and 111. Though it has been designated as structural since long, the exact functions, as well as the question which structural property it maintains, are unclear. To investigate the contribution of each of the second zinc ligands to the overall conformation, we have performed in vitro mutagenesis of class I and III ADH (Jelokova et al., 1994). The ligands were mutated, in separate constructs, to non-zinc liganding counterparts, Ala or Ser. Proteins expressed were found to be labile and therefore, were detectable only from crude extracts upon Western blot analysis. Confirmation of correctly working transcription processes was ascertained by positive Northern blot analyses. The recently published crystal structure of glucose dehydrogenase from the archaeon Thermoplasma acidophilum, reveals structural homology (in spite of low sequence identity) to ADH from horse liver and SDH from sheep liver (John et al., 1994). Glucose dehydrogenase is a tetramer and posesses a structural zinc atom, contained within a loop similar to that of ADH. However, the orientation of this structural loop with respect to the subunit is markedly different from that of ADH. This further illustrates the role of the zinc loop in the quaternary structures of several of the enzymes within the medium- chain dehydrogenase/reductase super-family, MDR (Persson et al. 1994)