The three-dimensional structures of dogfish M4 (muscle) and pig H4 (heart) lactate dehydrogenase (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) have been determined and correlated with the amino acid sequences of the dogfish M4, pig M4, pig H4, chicken M4, and chicken H4 lactate dehydrogenase isozymes. These results have been related to the known differences of physicochemical properties between the M and H lactate dehydrogenase isozymes. By far the largest structural alterations occur in the transition between the "apo" and "ternary complex" conformational states of the enzyme rather than between species or isozymes. The major catalytic difference can be explained by a replacement of alanine (in the M chain) with a glutamine (in the H chain) in the vicinity of the binding site of the coenzyme phosphates. The known immunological differentiation of the M and H isozymes is consistent with the differences in their amino acid sequences.The subunits of lactate dehydrogenase (LDH; L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) exist as two major structural forms, usually referred to as M (muscle) and H (heart) or A and B, respectively, which give rise to five different isozymes of the tetrameric molecule (1, 2) in higher vertebrates. The differences in the properties of the LDH isozymes are dependent on their subunit composition and are most exaggerated between the homotetramers M4 (LDH-5) and H4 (LDH-1). The most important of these differences are (independent of species): (i) The turnover number of the M4 isozyme is generally about twice that for the H4 isozyme (3-5).(ii) The activity of the H4 isozyme is more readily diminished by modification of the carboxyamide group on the 3 position of the nicotinamide (6). For instance, acetylpyridine adenine dinucleotide reduces the rate of catalysis by at least five when compared to NAD in the H4 enzyme, whereas the M4 isozyme is not especially sensitive to this difference of the NAD molecule (7). (iMi) The forward reaction lactate-to-pyruvate is inhibited to a far greater extent by high concentrations of pyruvate in the H4 than in the M4 isozyme (5, 7-9). (iv) Both the enzymic activity and the affinity of the substrate for enzyme are reduced to a greater extent in the M4 isozyme as the length of the aliphatic chain of the a-keto acid increases (10-12). (v) The H4 isozyme binds oxidized or reduced coenzyme better than the M4 isozyme (13-20). (vi) An antiserum induced in rabbit against the M4 isozyme of one species will crossreact with the M4 isozyme from several closely related species but not with the H4 isozyme of the same species (4,(21)(22)(23)(24)(25). Some appreciation of these differences is now possible from a correlation of recent studies on three-dimensional and primary structures of the H4 and M4 LDH isozymes.The tertiary structure of dogfish M4 LDH has been determined independently for the apo-enzyme and for a complex of LDH, coenzyme, and substrate analogue. The apo-enzyme has been studied at 2.0-A resolution, while a series of isomorphous ternary complexes h...

Comparisons have been made between the active center geometries of lactate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase, chymotrypsin and papain, and glyceraldehyde-3-phosphate dehydrogenase and papain. In the dehydrogenases, orientation of the nicotinamide ring about the glycosidic bond is determined by the substrate stereochemistry. The proper positioning of the carboxyamide moiety allows for the close approach of the C4 atom on the nicotinamide and the reactive carbon of the substrate. It follows that, once the conformation of the substrate or substrate intermediate has been established with respect to the functional groups in the enzyme, the A- or B-side specificity of the nicotinamide ring is predetermined. Hence, dehydrogenases which are divergently evolving from a common precursor must maintain the nicotinamide specificity if the protein fold of the catalytic domain is conserved. The tetrahedral intermediates produced during acylation of chymotrypsin and papain are found to be of opposite hand, while those of papain and glyceraldehyde-3-phosphate dehydrogenase can be regarded to be of the same hand. Thus the serine proteases, subtilisin and those of the chymotrypsin family, are of one hand while the cysteine enzymes, glyceraldehyde-3-phosphate dehydrogenase and papain, are of the other.

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