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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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