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Structural and Functional Similarities Between Mitochondrial Proc. Nat. Acad. Sci. USA Vol. 71, No. 4, pp. 1334-1338, April 1974 Structural and Functional Similarities Between Mitochondrial Malate Dehydrogenase and L-3-Hydroxyacyl CoA Dehydrogenase (subunit structure/immunological cross-reactivity/amino-acid composition/ specificity of hydrogen transfer/evolution) BARBARA E. NOYES*, BEAT E. GLATTHAAR, JOHN S. GARAVELLI, AND RALPH A. BRADSHAWf Division of Biology. and Biomedical Sciences, Department of Biological Chemistry, Washington University School of Medicine, St. Louis, Missouri 63110 Communicated by P. Roy Vagelos, December 27, 1973 ABSTRACT Pig heart mitochondrial malate dehy- In view of these findings, it is of interest to examine the drogenase (EC 1.1.1.37), which has been obtained free of electrophoretic subforms, has been shown to have a structural relationships of mitochondrial dehydrogenases, molecular weight of67,000 and to be composed of two poly- both among themselves and with the cytoplasmic forms. peptide chains. Comparison of these and other properties, Toward this end, the mitochondrial enzymes L-3-hydroxyacyl such as amino-acid composition, isoelectric point, and CoA dehydrogenase (EC 1.1.1.35) and malate dehydroge- keto-substrate inhibition, with those of L-3-hydroxyaeyl nase (EC 1.1.1.37) have been isolated in preparative amounts, CoA dehydrogenase (EC 1.1.1.35), another NAD+-depen- dent dehydrogenase of mitochondrial origin, suggests entirely free of undefined subforms, and their molecular prop- structural similarities of the type associated with pro- erties compared. From these results, it is concluded that these teins possessing common evolutionary origins. This con- enzymes probably possess the same order of structural simi- clusion is supported by immunological crossreactivity. larity that has been observed for the cytoplasmic malate and In view of these observations, the dissimilarity in the lactate dehydrogenases. stereospecificity of hydrogen transfer from cofactor to substrate catalyzed by the two enzymes is attributed to EXPERIMENTAL PROCEDURE 1800 rotation-in the binding orientation of the nicotinam- ide moiety of the NAD+, rather than to gross differences Homogeneous L-3-hydroxyacyl CoA dehydrogenase and mito- in the geometry of the active site of the two enzymes. chondrial malate dehydrogenase from pig heart muscle were prepared as described previously (13, 14). In each case, the Comparisons of the structure-function relationships of numer- principal component was isolated free of the more acidic sub- ous proteins have provided a clearly emerging picture of the forms that have been present in the preparations of other evolution of biological function within a class of proteins workers. Amino acid analyses, N-terminal analyses, sedi- through selective genetic alterations leading to local changes in side chain character and environment without appreciably affecting the conformation of the polypeptide backbone. TABLE 1. Amino acid composition of pig heart The family of serine proteases, where similarities in primary mitochondrial matate dehydrogenase* and three-dimensional structure are manifested in a variety of proteolytic enzymes with differing specificities from diverse Gregory phylogenetic origins (1-5), is the best documented example of Thorne Anderton et al. This this phenomenon. Altho~h less well documented at the three- (19) (20) (21) study dimensional level, Iyaouyme and a-lactalbumin (6, 7) and ex- Lysine 54.3 48.8 48.6 51.6 nerve growth factor and insulin (8) represent additional Histidine 12.7 12.4 14.1 10.5 amples of structurally related proteins with unique functions. Arginine 14.7 20.0 14.6 16.5 More recent investigations have revealed that an entirely Aspartic acid 46.9 48.8 47.5 50.1 parallel situation exists for the intracellular cytoplasmic de- Threonine 44.2 35.4 37.1 42.1 hydrogenases. Hill et al. (9), and Adams et al. (10) have dem- Serine 36.8 38.3 36.4 36.8 onstrated by means of x-ray crystallographic techniques that Glutamic acid 46.3 45.0 46.6 49.8 cytoplasmic malate dehydrogenase and lactate dehydrogenase Proline 50.3 33.5 43.1 46.4 possess subunits of very similar polypeptide conformation. Glycine 59.0 54.5 54.4 57.8 In addition, it has been shown that the amino-terminal por- Alanine 71.0 54.5 60.9 65.6 tions of these which contain the NAD + cofactor Cysteine 14.7 14.4 14.7 13.8 molecules, Valine 47.6 43.0 56.0 53.6 binding site, are closely related in conformation to the cor- Methionine 10.7 7.7 11.9 11.3 responding segments of glyceraldehyde-3-phosphate dehydro- Isoleucine 38.9 38.2 42.1 42.3 genase (11) and alcohol dehydrogenase (12), suggesting an Leucine 55.6 52.6 53.4 56.5 even more distant evolutionary relationship involving all four Tyrosine 9.4 9.2 9.3 9.9 enzymes. Phenylalanine 21.5 23.0 20.2 22.3 Tryptophan - 1.6 *Present address: Department of Biochemistry, Stanford Uni- Total 634.6 580.9 610.9 636.9 versity School of Medicine, Palo Alto, Calif. 94305. t To whom correspondence should be addressed. * Residues/67,000 daltons of protein. 1334 Downloaded by guest on September 28, 2021 Proc. Nat. Acad. Sci. USA 71 (1974) Comparison of Mitochondrial Dehydrogenases i335 mentation equilibrium analyses, and isoelectric focusing were Immunological Cross-Reactivity of 'Mitochondrial Malate performed as described previously (13, 15). Dehydrogenase and i-3-Hydroxyacyl CoA Dehydrogenase. Rabbit antibodies against the enzymes were produced by The results of quantitative precipitin analyses of L-3-hy- injecting 0.25 ml of 2 mg/ml of samples of L-3-hydroxyacyl droxyacyl CoA dehydrogenase and mitochondrial malate CoA dehydrogenase or mitochondrial malate dehydrogenase, dehydrogenase, presented in Fig. 2, illustrate cross-reactivity in complete Freund's adjuvant (DIFCO), into all four foot- between the two pig heart dehydrogenases. In the case of anti- pads of the animal. Two rabbits were injected with each pro- serum to L-3-hydroxyacyl CoA dehydrogenase, mitochondrial tein. Five weeks after the initial injection, an additional mg of malate dehydrogenase can precipitate about 2% of the anti- protein in incomplete Freund's adjuvant was injected into body at the equivalence point. L-3-Hydroxyacyl CoA de- each rabbit either subcutaneously or in the footpads. One hydrogenase, on the other hand, precipitated 11-12% of the week after the booster injection, the rabbits were bled from antibody in the antimitochondrial malate dehydrogenase the marginal ear vein on three successive days. Bleeding was serum at the equivalence. Cross-reactivity was observed with repeated on three successive days of each week until the anti- sera from all four rabbits. In control experiments utilizing body titer of the serum dropped. The serum was collected nonspecific rabbit sera, mitochondrial malate dehydrogenase after centrifugation of the clotted blood and stored at -20°. and L-3-hydroxyacyl CoA dehydrogenase precipitated less Quantitative precipitin tests were performed as described by than 10% and 20% the amount of protein precipitated from Kabat and Mayer (16). Total protein in the precipitate was the heterologous antiserum. In addition, cytoplasmic malate determined using the biuret reaction (17). dehydrogenase did not precipitate significant amounts of pro- The stereospecificity of hydrogen transfer was determined tein from either antiserum. for both enzymes using the procedures of Davies et al. (18). Stereospecificity of Transfer Catalyzed by Mito- [4-3H]NAD+ was a product of New England Nuclear Corp. Hydrogen chondrial Malate Dehydrogenase and L-3-Hydroxyacyl CoA and yeast alcohol dehydrogenase was purchased from Sigma Chemical Co. Dehydrogenase. Enzyme-catalyzed transfer of hydrogen be- tween substrate and cofactor has been reported to be stereo- RESULTS specific for the A-side (pro-R hydrogen) of the nicotinamide Characterization of the Principal Component of Pig Heart ring for mitochondrial malate dehydrogenase and specific for Mitochondrial Malate Dehydrogenase: Amino-Acid Composi- the B-side (pro-S hydrogen) of the ring for L-3-hydroxyacyl tion. The amino-acid composition of the principal mitochon- CoA dehydrogenase (18, 28). However, in each case, the mea- drial malate dehydrogenase of pig heart is summarized in surements were performed with impure enzyme. Consequently, Table 1, along with other compositions previously reported. the experiments were repeated with the homogeneous prepara- In each case, the values have been normalized to 67,000 tions known to be free of all subforms. [4-3H]NADH syn- molecular weight to facilitate comparison. With the exception thesized from [4-'H]NAD+ in the presence of ethanol and of the composition reported by Anderton (20), the values ob- yeast alcohol dehydrogenase is labeled on the B-side of the tained with the homogeneous protein (column 4) are in close nicotinamide ring, since alcohol dehydrogenase stereospecif- agreement with the preparations that also contain the more ically transfers the hydrogen from unlabeled ethanol to the acidic subforms (columns 1 and 3). This finding is consistent A-side of the ring (29). In subsequent conversion of the tri- with previous observations (14, 22). tiated NADH to NAD+ by L-3-hydroxyacyl CoA dehydro- genase or mitochondrial malate dehydrogenase, the tritium Molecular Weight. The molecular weight of the principal should remain in the NAD+ if the enzyme is A-specific, but component of mitochondrial malate dehydrogenase was deter- should be transferred to the substrate if the enzyme is B- mined by sedimentation equilibrium, in the presence and specific. In the case of mitochondrial malate dehydrogenase, absence of denaturing solvents, gel filtration, and sodium 99% of the initial radioactivity was accounted for in the dodecyl sulfate electrophoresis. The values obtained for the native protein are in excellent agreement with previously re- TABLE 2. Molecular weight data for pig heart ported numbers (Table 2). The subunit molecular weights, mitochondrial dehydrogenases which have not been previously reported, are consistent with a dimeric structure composed of polypeptide chains of equal MW native enzyme' size.
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