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Although essential for most forms of life, too much iron is harmful. To cope with these antagonistic phenomena an iron-storage molecule, ferritin, has evolved. The structure of horse spleen apoferritin, which has recently been refined, consists of 24 symmetrically related subunits forming a near-spherical hollow shell. In ferritin the central cavity is occupied by an iron core of 'ferrihydrite', a geologically ephemeral mineral found in hot or cold springs and in mine workings, or produced in the laboratory by heating solutions of ferric salts. Ferritin itself forms most readily from apoferritin, in the presence of dioxygen, from FeII, not FeIII. Access to its interior is through small intersubunit channels, and the protein influences both the rate of FeII-oxidation and the form of oxide produced.

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X-ray diffraction studies to 2.8A resolution have yielded the three-dimensional structure of mitochondrial aspartate aminotransferase (L-aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1), an isologous a2 dimer (M, = 2 X 45,000) The subunits are rich in secondary structure and contain two domains, one of which anchors the coenzyme, pyridoxal 5'-phosphate. Each active site lies between the subunits and is composed of residues from both of them.Aspartate aminotransferase (AATase; L-aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1) is the most studied of the vitamin B-6-dependent enzymes (1, 2). These enzymes catalyze a wide variety of transformations in amino acid metabolism. AATase effects the reversible transfer of an amino group from L-aspartate or L-glutamate to the a-keto acids a-ketoglutarate and oxalacetate. In the course of the double displacement reaction (3, 4) the coenzyme shuttles between the pyridoxal-P form (bound via an aldimine linkage to the E-amino group of a lysine) and the pyridoxamine-P form. "Syncatalytic" conformational changes occur in the enzyme matrix (5-7). Various coenzyme-substrate intermediates are identified by characteristic absorption and circular dichroism spectra (2, 8). The stereochemistry of pyridoxyl-catalyzed reactions has been probed (9), and a dynamic reaction mecha'nism has been proposed (10).Two homologous, genetically independent isozymes of AATase have been found in animal tissues, one in the cytosol (cAATase), the other in the mitochondrial matrix (mAATase). Both are a2 dimers of about 2 X 400 amino acids. The amino acid sequences of the pig (11-13) and chicken (refs. 14 and 15; U. Hausner, K. J. Wilson, and P. Christen, personal communication) isozymes are known or have been almost completely elucidated. Crystallized AATase offers an exceptional opportunity for the study of interactions amongst protein, coenzyme, and substrates during catalysis. The crystalline enzyme is catalytically competent (16). Single-crystal microspectrophotometric studies on cAATase (17, 18) and mAATase (16,19) have permitted the recognition of several reaction intermediates and the evaluation of some dissociation constants and kinetic parameters (19). Now a detailed picture of the enzyme is emerging from X-ray studies of such crystals. The high-resolution X-ray analyses of cAATase from chicken (20) and pig (21) are near completion. Here we report the three-dimensional structure of chicken mAATase in the internal aldimine form (pyridoxal-5'-P-enzyme) as revealed by a 2.8-A resolution X-ray study. RESULTS Structure determinationChicken mAATase crystallizes in a triclinic unit cell that contains one a2 dimer (22) whose subunits are related by a noncrystallographic dyad (23). Diffraction data to 2.8-A resolution of the native protein and the derivatives listed in Table 1 were collected on a CAD4F diffractometer (Enraf-Nonius, Delft, Netherlands). Previously found heavy atom sites (23) were reevaluated by difference Fourier maps. They were refined by alternate cycles of multiple i...

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The binding of coenzyme and substrate are considered in relation to the known primary and tertiary structure of lactate dehydrogenase (EC 1,1.1.27). The adenine binds in a hydrophobic crevice, and the two coenzyme phosphates are oriented by interactions with the protein. The positively charged guanidinium group of arginine 101 then folds over the negatively charged phosphates, collapsing the loop region overtthe active center and positioning. the ulreactive B side of the nicoti namide in a hydrophobic protein environment. Collapse of the loop also introduces various charged groups into the vicinity of the substrate binding site. The substrate is situated between histidine 195 and the C4 position on the nicotiriamide ring, and is partially oriented by interactions between its carboxyl group and arginine 171. The spatial arrangements of these groups may provide the specificity for the L-isomer of lactate.In this paper coenzyme and substrate binding to dogfish (Squalus acanthius) M4 lactate dehydrogenase (LDH; EC 1.1.1.27) will be discussed in relation to the known amino-acid sequence, the crystal structure determinations, and the effect of various chemical modifications of the enzyme and coenzyme. A comparison of the preliminary 3.0-A resolution structure of the abortive LDH: NAD-pyruvate ternary complex (1) with the more complete 2.0-A resolution structure of the apoenzyme provides information on possible conformational changes during catalysis. Everse and Kaplan (4) have recently reviewed many of the properties of LDH. Evidence from kinetic data indicates that there is an obligatory binding order of coenzyme followed by substrate (Fig. 1), at least near neutral pH (6-8). McPherson (9) has presented evidence to show that the adenine moiety of the coenzyme is required for binding of the nicotinamide moiety.Coenzyme binding Studies on the conformations of adenosine, AMP, and ADP at 2.8-A resolution and of NAD+ at 5.0-resolution, when diffused into crystals of the apoenzyme, are discussed by Chandrasekhar et al. (10). Diffraction patterns of the NADH binary complex closely resemble those of the NAD+ binary complex. Although the structure of each of these binary complexes differs slightly from the other, as a class, their mode of binding of the coenzyme to the apoenzyme is distinct from that of the coenzyme in the ternary complex (Fig. 2). Fig. 3 demonstrates this by a comparison of the structure of NAD in the ternary complex (in black) with (a) NAD+ and (b) AMP in binary complexes. The protein conformation of the apoenzyme differs markedly from that of the ternary complex structure in that the loop (residues 98-114) has folded down over the active center pocket in the ternary complexes. Many smaller conformational changes within the protein are associated with the large movement of the loop and the different position and conformation of the coenzyme.The adenosine binds in a hydrophobic crevice lined by valine 27, glycine 28, an alanine, glycine and valine in the region 29-33, valine 52, valine 54, methionin...

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