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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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Some time ago Schultz and his colleagues (Schultz et al., 1972; demonstrated that myeloperoxidase, when injected daily into tumour-bearing mice in conjunction with thio-TEPA, causes a significant decrease in the rate of tumour growth. Neither myeloperoxidase nor thio-TEPA alone appeared to be effective, and retardation in tumour growth was only observed as long as treatment was continued.In vitro, myeloperoxidase in the presence of hydrogen peroxide and a halide ion exerts a potent cytotoxic activity against a variety of cell types. These include bacteria (Klebanoff & Luebke, 1965;Klebanoff et al., 1966Klebanoff et al., , 1970Klebanoff, 1967Klebanoff, , 1968McRipley & Sbarra, 1967), fungi (Lehrer, 1969;Lehrer & Jan, 1970;Diamond et al., 1972;Howard, 1973), viruses (Belding et al., 1970, and a variety of mammalian cells (Klebanoff & Smith, 1970;Edelson & Cohn, 1973;Clark et al., 1975;Clark et al., 1976;Clark & Klebanoff, 1977). Presently available evidence suggests that the toxic activity of myeloperoxidase involves the generation of oxygen radicals or similar highly reactive species (Klebanoff & Clark, 1978). Other peroxidases such as lactoperoxidase and horseradish peroxidase have similar cytotoxic properties (Oram and Reiter, 1966;Jacques et al., 1975;Reiter, 1978 In order to construct an enzyme system that would function as the peroxidase-H202-halide system in vivo we immobilized the peroxidase together with a hydrogen peroxide producing enzyme onto small solid beads. This would assure that hydrogen peroxide is continually produced in the immediate vicinity of the peroxidase. Since chloride ions are ubiquitiously distributed, no special action was considered necessary to assure the availability of halide ions. Thus, a 20g CNBractivated Sepharose-4 was suspended in 50 ml 0.1 M phosphate buffer, pH 7.0, containing 1% lysine, and the suspension was stirred overnight. The gel was then thoroughly washed with water and stirred for 10h in 5% NaCl. The gel was again washed and then filtered over a Buchner funnel. The packed gel was resuspended in 20 ml buffer and 2ml 50% glutaraldehyde was added. After 1 h stirring at room temp. the gel was washed thoroughly with buffer. The gel was then added to a solution containing 20mg horseradish peroxidase (Sigma, type VI) and 4.5 ml glucose oxidase (A. niger; Sigma, type V, 1000 U ml-1). The mixture was gently shaken overnight at 4°C and the immobilized enzymes were separated from any unbound enzymes by the procedure described by Mosbach and Mattiassen (1976). The gel was then lyophilized to dryness and the dry powder was stored at -20°until use.Our first experiments were done using Novikoff hepatoma bearing rats. Three week old SpragueDawley rats were inoculated i.p. with 0.5ml of a Novikoff hepatoma cell suspension and the tumour was allowed to develop for 5 days. Each rat was then injected i.p. with 5mg of the immobilized enzymes, suspended in 1 ml PBS containing 0.1% glucose. This treatment was repeated for 5 consecutive days. All 10 control animals died within 15 days aft...

were equal, as were those of the enzymes from several microbial species; however, significantly smaller elution volumes, corresponding to higher molecular weights, were obtained for malate dehydrogenases of certain Gram-positive bacteria in the order Eubacteriales. Crystalline proteins, typical of small and large forms of malate dehydrogenase, were dissociated into enzymatically inactive subunits by treatment with acid, urea, or guanidine -HC1; partial reactivation was obtained by dialysis or dilution of the dissociating agent. ular sizes of proteins with molecular weights as high as 300,000.

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