The primary structure of glycolate oxidase from spinach has been determined. Six different types of peptide digest were investigated, utilizing CNBr, proteolytic enzymes, and chemical modifications to change a specificity of cleavage. In total, 90 peptides were purified and analyzed. The studies were aimed at correlation with crystallographic analysis of the same protein carried through in parallel and with cDNA studies which utilized initially determined amino acid sequences for synthesis of oligonucleotide probes. Continuous comparisons with the results from the crystallographic studies helped at an early stage to secure peptide overlaps, at the same time as the peptide data secured residue assignments in the electron density maps. In the end, all data agree and regions from all parts of the molecule have been checked by independent methods of analysis. The primary structure establishes the type of N-terminal post-translational processing, and yields information on segments not fully defined in electron density maps. Combined, the chemical, crystallographic, and cDNA data give extensive reliability. The peptide analysis shows that the N-terminus is blocked by acylation of the initiator methionine, which is in a primary structure typical for non-removal of the methionine in the processing events of the nascent protein chain. The molecule is comparatively rich in menthionine and some other generally less common residues, but has only one cysteine residue and no extensive hydrophobic segment. An amino acid sequence homology with flavocytochrome b2 from yeast, as expected from known similarities in tertiary structure, is observed (33% residue identities).

The crystal structure analysis of horse liver alcohol dehydrogenase has been extended to 2.4 Å resolution. From the corresponding electron density map of the apoenzyme we have determined the positions of the 374 amino acids in the polypeptide chain of each subunit.

The conformation of the polypeptide chain in horse liver alcohol dehydrogenase (EC 1.1.1.1), as well as the binding sites for some inhibitor molecules, have been determined from x-ray crystallographic data to a resolution of 2.9 A. Each subunit of the dimeric molecule is organized into two parts unequal in size and separated by a wide and deep active-site cleft. The adenosine moiety of the coenzyme is bound within the smaller region. Interactions between these coenzyme-binding substructures define the subunit contact area of the molecule. The "catalytic" zinc atoms are bound at the bottom of the clefts about 20 A from the surface of the molecule. The coenzyme binding region has a main-chain conformation very similar to a corresponding region in lactate and malate dehydrogenase. It is suggested that this substructure is a general one for binding of nucleotides and, in particular, the coenzyme NAD(+).

The structure at 5 å resolution of horse-liver alcohol dehydrogenase isoenzyme EE has been obtained from X-ray data from native protein crystals and crystals of three isomorphous derivatives. These crystals all have space-group C2221 with four molecules per unit cell: a = 56.0 å, b = 75.0 å and c = 181.4 å. Data to 2.9 å resolution were collected on precession photographs which were measured on a computer-controlled drum film scanner. Data to 5 å resolution were extracted from this data-set and used to compute an electron-density map. Values of E, an estimated error in FH for each derivative (and, in parentheses, values of fH = root mean square heavy-atom structure factor), are 57 (142) for the platinumcyanide derivative, 46 (134) for the goldcyanide and 42 (116) electrons for the double derivative substituted at both the platinum and the gold site. Corresponding R-values (R=Σ|FHP-|FP+fH|/ΣfH) were 0.33, 0.29 and 0.26. The average figure of merit was 0.79. A two-fold crystallographic axis relates the two identical subunits of the dimeric molecule. The molecular shape (approximately 45 × 55 × 110 å), the location of one zinc-atom per subunit and the probable location of the coenzyme binding site are presented. The zinc-atom is about 30 å away from the coenzyme binding site.

Horse liver alcohol dehydrogenase (LADH) catalyses the oxidation of alcohol to aldehyde when in the presence of the co-factor nicotinamide adenine dinucleotide. It is one of the many NAD-dependent dehydrogenases which are composed of two or four subunits each of molecular weight between 35.000 and 40.000. Of these a low resolution X-ray study has been reported for lactate dehydrogenase (l) and preliminary X-ray data for glyceraldehyde-3-phosphate dehydrogenase (2), soluble malate dehydrogenase (3) and liver alcohol dehydrogenase(4).