Inhibitor binding to the binuclear active site of tyrosinase: temperature, pH, and solvent deuterium isotope effects.

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.

Agaricus bisporus metapotyrosinase: preparation, characterization, and conversion to mixed-metal derivatives of the binuclear site.

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)

Benzoic acid inhibition of the alpha, beta, and gamma isozymes of Agaricus bisporus tyrosinase.

Inhibition of the catecholase and cresolase reactions of the alpha, beta, and gamma isozymes of Agaricus bisporus tyrosinase by benzoic acid was investigated at 25.0 and 8.0 degrees C at pH 5.60 in air-saturated solutions. Benzoic acid is a simple competitive inhibitor of the cresolase reaction of all three isozymes. In the catecholase reaction, however, benzoic acid is a partial uncompetitive inhibitor of the alpha and beta isozymes and a simple competitive inhibitor of gamma-tyrosinase. Equilibrium dialysis experiments, conducted under identical conditions to the kinetic studies, indicate that benzoic acid can bind to the alpha and gamma isozymes in the absence of organic substrate. The dissociation constants obtained by equilibrium dialysis are in good agreement with the kinetic Ki values determined from inhibition studies. Maximum binding of benzoic acid to alpha and gamma tyrosinase, however, is significantly less than one mole per mole of active sites. A scheme in which benzoic acid binds to the oxy-form of tyrosinase is proposed to account for the kinetic and equilibrium results.

Instability of purified 9-kDa porcine intestinal calcium-binding protein in the absence of calcium.

Purified 9-kDa porcine intestinal calcium-binding protein (ICaBP, Calbindin D9K) is unstable when stored at 4 degrees C in the absence of Ca(II). Cleavage of the polypeptide occurs producing approximately 5.2- and 3.7-kDa fragments. The former dimerizes giving a species which migrates on sodium dodecylsulfate-urea gels with an Mr 13,700, in contrast to the observed Mr 11,000 for native ICaBP. The fragmentation also results in an irreversible loss of high affinity Ca(II) bound to ICaBP prevents fragmentation. The dimer can be isolated from aged preparations of apo-ICaBP and is stable on further storage with or without Ca(II) present. The observed molecular weights of the fragments along with the amino acid analysis and ultraviolet spectra of the dimer suggest cleavage of the polypeptide chain of ICaBP in the vicinity of residue 49.

Synthesis and 1H NMR spectroscopic characterization of trans-[Pt(NH3)2[d(ApGpGpCpCpT)-N7-A(1),N7-G(3)]].

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.

Photoinactivation of agaricus bisporus tyrosinase: modification of the binuclear copper site.

Irradiation of Agaricus bisporus tyrosinase in the presence of citrate at pH 5.6 with 300-420-nm light results in a loss of both catecholase activity and cresolase activity. The light-sensitive species appears to be an enzyme-citrate complex, most likely involving coordination of citrate to the active site copper. One copper ion from each binuclear active site can be removed from the inactivated enzyme, resulting in the formation of a met apo derivative. The electron spin resonance spectrum of met apo tyrosinase resembles that of met apo hemocyanin and half-met Neurospora tyrosinase. It is consistent with a distorted square-planar geometry around the copper and with either nitrogen or nitrogen and oxygen ligands. Amino acid analysis indicates that four histidines on the heavy subunit are destroyed during the inactivation process. Some or all of these histidines may serve as ligands to the copper ion which becomes labile after inactivation. Photoinactivation results in decarboxylation of citrate and does not require the presence of oxygen. The reaction may involve generation of a free radical from the citrate which then attacks nearby histidine residues.

Anion binding to the four-copper form of bovine erythrocyte superoxide dismutase: Mechanistic implications.

The binding of thiocyanate, azide, and other anions to superoxide dismutase (SOD) containing copper in both the copper and zinc sites of the native enzyme (Cu2Cu2SOD) has been studied. Electron spin resonance spectroscopy was used to show that binding of SCN- to Cu2Cu2SOD breaks the imidazolate bridge between the two copper centers. Thiocyanate displaces the bridging histidine from the Cu2+ ion in the copper site and also replaces the aspartic acid ligand from the Cu2+ ion in zinc site. This conclusion is supported by studies with Ag2Cu2SOD, where silver is in the native copper site and copper is in the native zinc site. At low concentrations, N3- ion displaces axially coordinated water from Cu2Cu2SOD. At higher concentrations, N3- also breaks the imidazolate bridge. Parallel behavior was observed for SCN- and N3- binding to a model compound for Cu2Cu2SOD, showing that the difference between the two anions is a consequence of their copper binding properties and is not due to secondary interactions with the protein active site. The SCN- complex of Cu2Cu2SOD is catalytically active, indicating that the intact imidazolate bridge is not essential to the mechanism of superoxide dismutase action. Phosphate ion breaks the histidine bridge in Cu2Cu2SOD, whereas cyanate, formate, and fluoride ions do not. At about pH 11, hydroxide ion promotes the irreversible formation of a copper complex of deprotonated peptide nitrogen atoms for Cu2Cu2SOD but not for Cu2Zn2SOD in the same pH range.

Tetrakis(acetoxymercuri)methane: a polymetallic reagent for labeling sulfur in nucleic acids.

Tetrakis(acetoxymercuri)methane binds to the sulfur atom of 6-thioguanosine and also to the 4-thiouridine residue of Escherichia coli tRNAVal. A 4:1 complex is formed between 6-thioguanosine and tetrakis(acetyoxymercuri)methane. Addition of 3 equivalents of N,N-dimethyl-2-amino-ethanethiol hydrochloride to tetrakis(acetoxymercuri)methane effectively blocks three of the four mercury atoms, rendering the compound monofunctional toward 6-thioguanosine. Under appropriate conditions, tetrakis(acetyoxymercuri)methane, in the presence or absence of N,N-dimethyl-2-amino-ethanethiol hydrochloride, binds to the 4-thiouridine residue in E. coli tRNAVal without forming intermolecular crosslinks. These results suggest that tetrakis(acetoxymercuri)methane will be a useful polymetallic reagent for labeling sulfur sites in polynucleotides. It may also prove to be a valuable reagent for preparing heavy metal derivatives of proteins for x-ray crystallographic study.

Platinum binds selectively to phosphorothioate groups in mono- and polynucleotides: a general method for heavy metal staining of specific nucleotides.

Platinum binding to nucleoside phosphorothioates has been examined to determine their suitability as heavy metal labeling sites for the potential electron microscopic sequencing of nucleic acids. The complex platinum terpyridine nitrate forms a 1:1 adduct with either adenosine or uridine monophosphorothioate. Spectroscopic evidence strongly indicates the presence of platinum-sulfur bonds. Both platinum terpyridine nitrate and chloroterpyridineplatinum(II) bind to poly(sA-U), a polymer prepared from adenosine 5'-O-(1-thiotriphosphate) and UTP. Binding to the sulfur atoms of the phosphorothioate groups is quantitative, as shown by double label experiments using [35S]poly(sA-U) and [3H]chloroterpyridine-platinum(II). Similar experiments with [14C]poly(A-U) indicated no platinum binding. No evidence of nicking or loss of sulfur from poly(sA-U) could be detected after platinum binding. The phosphorothioate group is a strong, highly selective binding site for platinum in polynucleotides. Previous studies have demonstrated quantitative enzymatic incorporation of phosphorothioate groups into a polynucleotide adjacent to a specific base [Matzura, H. & Eckstein, F. (1968) Eur. J. Biochem. 3, 448-452]. The use of heavy metal-labeled phosphorothioate groups for the sequencing of nucleic acids by electron microscopy therefore appears feasible.