Competitive inhibition of the monophenolase reaction of tyrosinase by a variety of compounds was investigated with respect to temperature and pH. Derivatives of benzoic acid as well as p-nitrophenol were the best inhibitors. Toluene and several N-heterocycles were all weak inhibitors. Thermodynamic parameters of toluene inhibition were qualitatively different from those of aromatic acids and were consistent with a hydrophobic binding site for toluene. Inhibition by both aromatic acids and p-nitrophenol was strongly pH-dependent over the range 5.1-8.0, with inhibitor binding favored at lower pH. In contrast, toluene binding and indazole binding were pH-independent while benzimidazole binding was favored at higher pH. For both carboxylic acids and p-nitrophenol, inhibitor binding was quantitatively accounted for by assuming the protonated, electrically neutral form of the inhibitor is the reactive species. A solvent deuterium isotope effect on the binding of benzoic acid was observed. A proton inventory study indicated that a single proton transferred from benzoic acid to a site with a fractionation factor of 0.64 +/- 0.02 accounts for the isotope effect. The identity of the proton acceptor and possible mechanistic implications are discussed.

Ligand-bridged polymetallic centers occur in numerous metalloproteins where they perform electron transfer, oxygen transport, and catalytic redox functions.1 The properties of a variety of proteins containing such centers are given in Table I.2-10 One of the most extensively studied members of this group, and probably the best understood, is the Cu-Zn superoxide dismutase (SOD+).Superoxide dismutases are a ubiquitous group of enzymes. Procaryotes and mitochondria of eucaryotic cells have ironor manganese-containing superoxide dismutases while a copper-and zinc-containing superoxide dismutase is found in the cytosol of eucaryotic cells.11-13 These enzymes are believed to protect cells from the toxic effects of superoxide ion, or some product derived from it, by catalyzing the dismutation reaction given in eq 1. Hydrogen peroxide formed in this re-202-+ 2H+ = 02 + H202(1) action is destroyed in vivo, for example, by catalase.Bovine erythrocyte SOD has a molecular weight of 31200 and is composed of two identical subunits.1415 Each subunit contains an active site having one copper and one zinc atom. The X-ray crystal structure at 3-A resolution has been published,2 and coordinates from 2-A data are available.16 As shown in Figure 1, the copper is coordinated to four imidazole nitrogen atoms from histidines-44, -46, -61, and -118 while the zinc is coordinated to histidines-61, -69, and -78 and the carboxylate group of aspartic acid-81. The most interesting and still unique feature of the active site structure is the imidazolate bridge between the two metal ions. The pyrrole hydrogen of the imidazole group of histidine-61 has been removed, allowing for coordination of both copper and zinc. The geometry aground the zinc is approximately tetrahedral, and the four protein ligands to the copper are distorted square planar. Proton NMR data indicate that a water molecule is coordinated to

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Quantitating the binding of ethidium bromide with DNA by monitoring the fluorescence of an ethidium-DNA solution.

The reaction of trans-[Pt(NH3)2Cl2] with the sodium salt of [d(ApGpGpCpCpT)]2 in aqueous solution at 37 degrees C was monitored by reversed-phase high-performance liquid chromatography and UV spectroscopy. Two intermediates, most likely monofunctional adducts, were observed, which subsequently formed one predominant single-stranded product, as well as several polymeric species proposed to be interstrand cross-linked products. The single-stranded adduct was structurally characterized by 1H NMR spectroscopy. From the pH dependence of the chemical shifts, two-dimensional homonuclear chemical shift correlation (COSY) spectroscopy, and one- and two-dimensional nuclear Overhauser effect (NOESY) experiments, the platinum(II) moiety was found to be coordinated to the N7 positions of adenine(1) and guanine(3), with the intervening guanine(2) base destacked from its neighboring residues. This intrastrand 1,3 adduct induces changes in the backbone torsion angles and causes the deoxyribose ring of adenine(1) to switch from a C2'-endo to a predominantly C3'-endo conformation. The other deoxyribose rings retain B DNA type conformations. The structure of trans-[Pt(NH3)2[d(ApGpGpCpCpT)-N7-A(1),N7-G(3)]] differs from those previously reported for cis-DDP 1,2- and 1,3-intrastrand oligonucleotide adducts but is consistent with the structures of trans-DDP 1,3-intrastrand adducts of two previously reported trinucleotides.