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

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

No abstract

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.

The alpha, beta, and gamma isozymes of Agaricus bisporus tyrosinase undergo inactivation in the presence of oxalate. The inactivation rate law is first order in enzyme and second order in oxalate. On a more rapid time scale than inactivation, oxalate acts as a competitive inhibitor of the catecholase reaction of tyrosinase. After removal of oxalate by dialysis, the inactivated enzyme is found to contain 50% of the original copper, all of which is present as paramagnetic, mononuclear copper sites. The ESR parameters of this copper indicate a tetragonal environment with nitrogen and oxygen ligands. The product of oxalate inactivation has lost one copper from each binuclear site and is thus a metapo derivative. Addition of Cu(II) to metapotyrosinase results in complete recovery of copper and catalytic activity. Prolonged storage of metapotyrosinase, in the absence of any additional Cu(II), results in copper migration, producing a 50% recovery of the original specific activity, expressed on a protein basis. Copper migration converts metapo sites into equal numbers of reconstituted, holo sites and fully apo sites. Both copper migration and copper reconstitution follow apparent first-order kinetics and are pH dependent. The involvement of two ionizable groups accounts for the observed pH dependence of each process. For copper migration pKa values of 6.0 and 8.8 were found, while for copper reconstitution the pKa values were 5.4 and 6.9. Addition of either Co(II) or Zn(II) to metapotyrosinase results in the formation of enzymatically inactive, mixed-metal derivatives of the binuclear copper site having one Cu(II) and one Co(II) or Zn(II) ion.(ABSTRACT TRUNCATED AT 250 WORDS)

No abstract

When exposed to either sodium dodecyl sulfate or guanidinium chloride, ascorbate oxidase (molecular weight 140,000, 8-10 copper atoms) dissociates into two copper-free subunits, each of about half the molecular weight of the native enzyme. Removal of the denaturant results in aggregation of the subunits and it has not been found possible to restore the copper and activity. All evidence indicates that the two subunits of molecular weight 65,000, comprising the native enzyme, are identical. Treatment of the native enzyme with sodium dodecyl sulfate and either 2-mercaptoethanol or 2mercaptoethylamine results in two new bands on sodium dodecyl sulfate electrophoresis. The components corresponding to these bands have molecular weights of 38,000 (A chain) and 28,000 (B chain). The same two components are obtained when disulfides of the native enzyme are cleaved with cyanide.