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Q-band electron nuclear double resonance (ENDOR) has measured anisotropic, distance-dependent dipolar hyperfine couplings from iron in ferric bleomycin [Fe(III)-BLM] and in activated bleomycin [Act-BLM] to 31 P of substrate DNA. Studies were focused on bleomycin complexes with a self-complementary duplex DNA 10-mer, d(GGAAGCTTCC) 2 , containing a 5′-G-C-3′ sequence that is selective for bleomycin cleavage (Mao, Q.; Fulmer, P.; Li, W.; DeRose, E. G.; Petering, D. H. J. Biol. Chem. 1996, 271, 6185-6191). Bleomycin complexes with high molecular weight calf thymus DNA were also used. Fe(III)-BLM and Act-BLM complexes with the 10-mer and the calf thymus DNA showed anisotropic 31 P dipolar hyperfine couplings from which an Fe(III)-to-31 P distance was estimated at 7.4 ( 0.2 Å. High-resolution, angle-selected ENDOR of the Fe(III)-BLM 10-mer complex showed that the Fe(III)-to-31 P vector lay at 25 ( 5°to the maximal g value direction, where the latter direction pointed near the exchangeable protons on axial BLM ligands and is associated with the maximal hyperfine couplings of these protons (Veselov, A.; Sun, H.; Sienkiewicz, A.; Taylor, H.; Burger, R. M.; Scholes, C. P. J. Am. Chem. Soc. 1995, 117, 7508-7512). Proton ENDOR features of the Fe(III)-BLM but not Act-BLM were perturbed by DNA substrate. In the presence of the 10-mer and the calf thymus DNA, proton ENDOR revealed distinct perturbation to the frequencies of three sets of nonexchangeable protons assigned to the BLM macrocycle and estimated to be 2.9-3.5 Å from Fe(III) and to the frequencies of exchangeable, axially located protons. In contrast, the hyperfine couplings of covalently bonded, first-shell nitrogen and [ 17 O]peroxy ligands were unchanged.

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5'-Nucleotidase prepared from muscle of small intesting of pig is strongly inhibited by nucleoside di- and triphosphates and their phosphonate analogs. Substrate kinetics appromate the Michaelis-Menten for for AMP, which shows a Km of 3-6 muM at pH 5.3-7.2. Inhibition is characterized as partial competitive, except at pH 5.3, where inhibition by ATP is noncompetitive. The Ki values for several inhibitors have been determined, and their departure from completeness of competitive inhibition has been studied. Inhibitor cooperativity of the type reported for the enzyme from sheep brain (P. L. Ipata (1968), Biochemistry 7, 507) was not observed for the enzyme from gut. In addition we failed to confirm sigmoid inhibition kinetics with 5'-nucleotidase from sheep brain.

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