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To ensure successful parasitism, parasitoid wasps inject venom along with their eggs into their hosts. The venom serves to suppress host immune responses, including melanization. Venom from Pteromalus puparum, a pupal endoparasitoid, inhibits melanization of host hemolymph in vitro in a dose-dependent manner. Using assay-guided fractionation, a serpin splicing isoform with phenoloxidase inhibitory activity was identified as P. puparum serpin-1, venom isoform (PpS1V). This serpin gene has 16 predicted splicing isoforms that differ only in the C-terminal region. RT-PCR results show that the specific serpin isoform is differentially expressed in the venom gland. Recombinant PpS1V (rPpS1V) suppresses host prophenoloxidase (PPO) activation rather than inhibiting the phenoloxidase directly. Pulldown assays show that PpS1V forms complexes with two host hemolymph proteins, here named Pieris rapae hemolymph proteinase 8 (PrHP8) and P. rapae prophenoloxidase-activating proteinase 1 (PrPAP1), based on gene sequence blasting and phylogenetic analysis. The role of rPrPAP1 in the PPO activation cascade and its interaction with rPpS1V were confirmed. The stoichiometry of inhibition of PrPAP1 by PpS1V is 2.3. PpS1V also inhibits PPO activation in a non-natural host, Ostrinia furnacalis, through forming a complex with O. furnacalis serine protease 13 (OfSP13), an ortholog to PrPAP1. Our results identify a venom-enriched serpin isoform in P. puparum that inhibits host PPO activation, probably by forming a complex with host hemolymph proteinase PrPAP1.

The melanization cascade, activated by the prophenoloxidase (proPO) system, plays a key role in the production of cytotoxic intermediates, as well as melanin products for microbial sequestration in invertebrates. Here, we show that the proPO system is an important component of the Penaeus monodon shrimp immune defense toward a major viral pathogen, white spot syndrome virus (WSSV). Gene silencing of PmproPO(s) resulted in increased cumulative shrimp mortality after WSSV infection, whereas incubation of WSSV with an in vitro melanization reaction prior to injection into shrimp significantly increased the shrimp survival rate. The hemolymph phenoloxidase (PO) activity of WSSV-infected shrimp was extremely reduced at days 2 and 3 post-injection compared with uninfected shrimp but was fully restored after the addition of exogenous trypsin, suggesting that WSSV probably inhibits the activity of some proteinases in the proPO cascade. Using yeast two-hybrid screening and co-immunoprecipitation assays, the viral protein WSSV453 was found to interact with the proPO-activating enzyme 2 (PmPPAE2) of P. monodon. Gene silencing of WSSV453 showed a significant increase of PO activity in WSSV-infected shrimp, whereas co-silencing of WSSV453 and PmPPAE2 did not, suggesting that silencing of WSSV453 partially restored the PO activity via PmPPAE2 in WSSV-infected shrimp. Moreover, the activation of PO activity in shrimp plasma by PmPPAE2 was significantly decreased by preincubation with recombinant WSSV453. These results suggest that the inhibition of the shrimp proPO system by WSSV partly occurs via the PmPPAE2-inhibiting activity of WSSV453.

Many insects eat the green leaves of plants but excrete black feces in an as yet unknown mechanism. Insects cannot avoid ingesting pathogens with food that will be specifically detected by the midgut immune system. However, just as in mammals, many pathogens can still escape the insect midgut immune system and arrive in the hindgut, where they are excreted out with the feces. Here we show that the melanization of hindgut content induced by prophenoloxidase, a key enzyme that induces the production of melanin around invaders and at wound sites, is the last line of immune defense to clear bacteria before feces excretion. We used the silkworm Bombyx mori as a model and found that prophenoloxidase produced by hindgut cells is secreted into the hindgut contents. Several experiments were done to clearly demonstrate that the blackening of the insect feces was due to activated phenoloxidase, which served to regulate the number of bacteria in the hindgut. Our analysis of the silkworm hindgut prophenoloxidase discloses the natural secret of why the phytophagous insect feces is black and provides insight into hindgut innate immunity, which is still rather unclear in mammals.

A serine proteinase pathway in insect hemolymph leads to prophenoloxidase activation, an innate immune response against pathogen infection. In the tobacco hornworm Manduca sexta, recombinant hemolymph proteinase 14 precursor (pro-HP14) interacts with peptidoglycan, autoactivates, and initiates the proteinase cascade (Ji, C., Wang, Y., Guo, X., Hartson, S., and Jiang, H. (2004) J. Biol. Chem. 279, 34101-34106). Here, we report the purification and characterization of pro-HP14 from the hemolymph of bacteria-injected M. sexta larvae. The zymogen, consisting of a single polypeptide with a molecular mass of 68.5 kDa, is truncated at the amino terminus. It is converted to a two-chain active form in the presence of beta-1,3-glucan (a fungal cell wall component) and beta-1,3-glucan recognition protein-2. The 45-kDa heavy chain contains four low-density lipoprotein receptor A repeats, one Sushi domain, and one unique cysteine-rich region, whereas the 30-kDa light chain contains a serine proteinase domain, which was labeled by [(3)H]diisopropyl fluorophosphate. Pro-HP14 in the plasma strongly binds curdlan, zymosan, and yeast and interacts with peptidoglycan and Micrococcus luteus. Addition of autoactivated HP14 elevated phenoloxidase activity level in the larval plasma. Recombinant M. sexta serpin-1I reduced prophenoloxidase activation by inhibiting HP14. These data are consistent with the current model on initiation and regulation of the prophenoloxidase activation cascade upon recognition of pathogen-associated molecular patterns by specific pattern recognition proteins.

The malaria vector Anopheles gambiae is capable of multiple immune responses against Plasmodium ookinetes. Accumulating evidence in several insect species suggests the involvement of serine protease cascades in the initiation and coordination of immune responses. We report molecular and reverse genetic characterization of two mosquito clip domain serine proteases, CLIPB14 and CLIPB15, which share structural similarity to proteases involved in prophenoloxidase activation in other insects. Both CLIPs are expressed in mosquito hemocytes and are transcriptionally induced by bacterial and Plasmodium challenges. Functional studies applying RNA interference revealed that both CLIPs are involved in the killing of Plasmodium ookinetes in Anopheles. Studies on parasite melanization demonstrated an additional role for CLIPB14 in the prophenoloxidase cascade. We further report that both CLIPs participate in defense toward Gram-negative bacteria. Our findings strongly suggest that clip domain serine proteases serve multiple functions and play distinctive roles in several immune pathways of A. gambiae.

One innate immune response pathway of insects is a serine protease cascade that activates prophenol oxi-dase (pro-PO) in plasma. However, details of this pathway are not well understood, including the number and order of proteases involved. Protease inhibitors from the serpin superfamily appear to regulate the proteases in the pathway. Manduca sexta serpin-4 and serpin-5 suppress pro-PO activation in plasma, apparently by inhibiting proteases upstream of the direct activator of pro-PO. To identify plasma proteases inhibited by these serpins, we used immunoaffinity chromatography with serpin antibodies to isolate serpin-protease complexes that formed after activation of the cascade by exposure of plasma to bacteria or lipopolysaccharide. Covalent complexes of serpin-4 with hemolymph proteases HP-1 and HP-6 appeared in plasma activated by Gram-positive or Gram-negative bacteria, whereas serpin-4 complexes with HP-21 and two unidentified proteases were unique to plasma treated with Gram-positive bacteria. HP-1 and HP-6 were also identified as target proteases of serpin-5, forming covalent complexes after bacterial activation of the cascade. These results suggest that HP-1 and HP-6 may be components of the pro-PO activation pathway, which are activated in response to infection and regulated by serpin-4 and serpin-5. HP-21 and two unidentified proteases may participate in a Gram-positive bacteria-specific branch of the pathway. Several plasma proteins that co-purified with serpin-protease complexes, most notably immulectins and serine protease homologs, are known to be components of the pro-PO activation pathway. Our results suggest that after activation by exposure to bacteria, components of the pro-PO pathway associate to form a large noncovalent complex, which localizes the melanization reaction to the surface of invading microorganisms.

The Cu2+ complexes of the 1-16 and the 1-20 fragments of the Alzheimer's disease-related beta-amyloid peptide (CuAbeta) show significant oxidative activities toward a catechol-like substrate trihydroxylbenzene and plasmid DNA cleavage. The latter reflects possible oxidative stress to biological macromolecules, yielding supporting data to the pathological role of these soluble Abeta fragments. The former exhibits enzyme-like kinetics and is dependent on [H2O2], exhibiting k(cat) of 0.066 s-1 (6000-fold higher than the reaction without CuAbeta) and k(cat)/Km of 37.2 m-1s-1 under saturating [H2O2] of approximately 0.24%. This kinetic profile is consistent with metal-centered redox chemistry for the action of CuAbeta. A mechanism is proposed by the use of the catalytic cycle of dinuclear catechol oxidase as a working model. Trihydroxylbenzene is also oxidized by CuAbeta aerobically without H2O2, affording rate constants of 6.50x10(-3) s-1 and 3.25 m-1s-1. This activity is also consistent with catechol oxidase action in the absence of H2O2, wherein the substrate binds and reduces the Cu2+ center first, followed by O2 binding to afford the mu-eta2:eta2-peroxo intermediate, which oxidizes a second substrate to complete the catalytic cycle. A tetragonally distorted octahedral metal coordination sphere with three coordinated His side chains and some specific H-bonding interactions is concluded from the electronic spectrum of CuAbeta, hyperfine-shifted 1H NMR spectrum of CoAbeta, and molecular mechanics calculations. The results presented here are expected to add further insight into the chemistry of metallo-Abeta, which may assist better understanding of the neuropathology of Alzheimer's disease.

A serine proteinase cascade in insect hemolymph mediates prophenoloxidase activation, a defense mechanism against pathogen or parasite infection. Little is known regarding its initiating proteinase or how this enzyme is activated in response to invading microorganisms. We have isolated from the tobacco hornworm, Manduca sexta, a cDNA encoding a modular protein designated hemolymph proteinase 14 (HP14). It contains five low density lipoprotein receptor class A repeats, a Sushi domain, a unique Cys-rich region, and a proteinase-catalytic domain. The HP14 mRNA exists in fat body and hemocytes of the naive larvae, and its level increases significantly at 24 h after a bacterial challenge. We expressed proHP14 with a carboxyl-terminal hexahistidine tag in a baculovirus/insect cell system and detected the recombinant protein in two forms. The 87-kDa protein was primarily intracellular, whereas the 75-kDa form was present in the medium. Interaction with peptidoglycan resulted in proteolytic processing of the purified zymogen and generation of an amidase activity. Supplementation of hemolymph with proHP14 greatly enhanced prophenoloxidase activation in response to Micrococcus luteus. These data suggest that proHP14 is a pattern recognition protein that binds to bacteria and autoactivates and triggers the prophenoloxidase activation system in the hemolymph of M. sexta.

The prophenoloxidase (proPO) cascade is a major innate immune response in invertebrates, which is triggered into its active form by elicitors, such as lipopolysaccharide, peptidoglycan, and 1,3-beta-D-glucan. A key question of the proPO system is how pattern recognition proteins recognize pathogenic microbes and subsequently activate the system. To investigate the biological function of 1,3-beta-D-glucan pattern recognition protein in the proPO cascade system, we isolated eight different 1,3-beta-D-glucan-binding proteins from the hemolymph of large beetle (Holotrichia diomphalia) larvae by using 1,3-beta-D-glucan immobilized column. Among them, a 20- and 17-kDa protein (referred to as Hd-PGRP-1 and Hd-PGRP-2) show high sequence identity with the short forms of peptidoglycan recognition proteins (PGRPs-S) from human and Drosophila melanogaster. To be able to characterize the biochemical properties of these two proteins, we expressed them in Drosophila S2 cells. Hd-PGRP-1 and Hd-PGRP-2 were found to specifically bind both 1,3-beta-D-glucan and peptidoglycan. By BIAcore analysis, the minimal 1,3-beta-D-glucan structure required for binding to Hd-PGRP-1 was found to be laminaritetraose. Hd-PGRP-1 increased serine protease activity upon binding to 1,3-beta-D-glucan and subsequently induced the phenoloxidase activity in the presence of both 1,3-beta-D-glucan and Ca(2+), but no phenoloxidase activity was elicited under the same conditions in the presence of peptidoglycan and Ca(2+). These results demonstrate that Hd-PGRP-1 can serve as a receptor for 1,3-beta-D-glucan in the insect proPO activation system.

Phenol oxidase, a copper-containing enzyme, is widely distributed not only in animals but also in plants and fungi, which is responsible for initiating the biosynthesis of melanin. Activation of prophenol oxidase in arthropods is important in host defense. However, the prophenol oxidase-activating system remains poorly understood at the molecular level. Here we show that the coagulation cascade of the horseshoe crab Tachypleus tridentatus is linked to prophenol oxidase activation, with the oxygen carrier hemocyanin functioning as a substitute for prophenol oxidase. Tachypleus clotting enzyme functionally transforms hemocyanin to phenol oxidase, and the conversion reaches a plateau at 1:1 stoichiometry without proteolytic cleavage. The active site-masked clotting enzyme also has the same effect, suggesting that complex formation of the clotting enzyme with hemocyanin is critical for the conversion. The two systems of blood coagulation and prophenol oxidase activation may have evolved from a common ancestral protease cascade.

Cell lines from the malaria vector Anopheles gambiae have been established as a tool for the study of the mosquito innate immune system in vitro. Here, we describe the first continuous insect cell line that produces prophenoloxidase (PPO). This cell line (4a-3B) expresses constitutively six PPO genes, three of which are novel (PPO4, PPO5, and PPO6). The PPO genes show distinct temporal expression profiles in the intact mosquito, spanning stages from the embryo to the adult in an overlapping manner. Transient induction of larva-specific PPO genes in blood-fed adult females suggests that the developmental hormone 20-hydroxyecdysone may be involved in PPO gene regulation. Indeed, exposure of 4a-3B cells to 20-hydroxyecdysone in culture results in induction of those PPO genes that are mainly expressed in early developmental stages, and repression of PPO5, which is preferentially expressed at the adult stage. The cell line shows bacteria-induced immune transcripts that encode defensin and Gram-negative bacteria-binding protein, but no induction of PPO transcripts. This cell line most likely derives from a hemocyte lineage, and represents an appropriate in vitro model for the study of the humoral and cellular immune defenses of A. gambiae.

Serpin gene-1 from the tobacco hornworm, Manduca sexta, encodes, through alternative exon usage, 12 reactive site variants (Jiang, H., Wang, Y. and Kanost, M. R., (1994) J. Biol. Chem. 269, 55-58; Jiang, H., Wang, Y., Huang, Y., Mulnix, A. B., Kadel, J., Cole, K., and Kanost, M. R. (1996) J. Biol. Chem. 271, 28017-28023). These 43-kDa proteins differ from each other only in their COOH-terminal 39-46 residues, which include the reactive site. To test the hypothesis that these proteins are proteinase inhibitors of diverse selectivities and to begin to elucidate their physiological functions, we expressed the 12 serpin-1 variants in Escherichia coli. Seven of the variants inhibited mammalian serine proteinases, with association rate constants comparable with those of human serpins. Serpin-1A, with a P1 Arg residue, inhibited both trypsin and plasmin. Serpin-1B (P1 Ala) and serpin-1F (P1 Val) inhibited porcine pancreatic elastase and human neutrophil elastase. Serpin-1H, -1K, and -1Z, all with a Tyr residue at the P1 position, inhibited chymotrypsin and cathepsin G. Serpin-1I (P1 Leu) inhibited both elastase and chymotrypsin. Nine of the serpin variants were active as inhibitors of microbial serine proteinases, including subtilisin Carlsberg, proteinase K, and two proteinases secreted by an entomopathogenic fungus, Metarhizium anisopliae. In addition, one of the serpin variants, serpin-1J, strongly inhibited activation of M. sexta hemolymph phenoloxidase, a pathway involving a serine proteinase cascade. This pathway is a component of the defensive response of insects to microbial infection. These results suggest that the products of M. sexta serpin gene-1 may be important in regulating both exogenous and endogenous serine proteinases in hemolymph.

The prophenoloxidase (proPO) system is activated upon recognition of pathogens by pattern recognition proteins (PRPs), including a lipopolysaccharide- and β-1,3-glucan-binding protein (LGBP). However, shrimp LGBPs that are involved in the proPO system have yet to be clarified. Here, we focus on characterizing the role of a Penaeus monodon LGBP (PmLGBP) in the proPO system. We found that PmLGBP transcripts are expressed primarily in the hemocytes and are increased at 24 h after pathogenic bacterium Vibrio harveyi challenge. The binding studies carried out using ELISA indicated that recombinant (r)PmLGBP binds to β-1,3-glucan and LPS with a dissociation constant of 6.86 × 10(-7) M and 3.55 × 10(-7) M, respectively. Furthermore, we found that rPmLGBP could enhance the phenoloxidase (PO) activity of hemocyte suspensions in the presence of LPS or β-1,3-glucan. Using dsRNA interference-mediated gene silencing assay, we further demonstrated that knockdown of PmLGBP in shrimp in vivo significantly decreased the PmLGBP transcript level but had no effect on the expression of the other immune genes tested, including shrimp antimicrobial peptides (AMPs). However, suppression of proPO expression down-regulated PmLGBP, proPO-activating enzyme (PmPPAE2), and AMPs (penaeidin and crustin). Such PmLGBP down-regulated shrimp showed significantly decreased total PO activity. We conclude that PmLGBP functions as a pattern recognition protein for LPS and β-1,3-glucan in the shrimp proPO activating system.

Tyrosinase (EC 1.14.18.1) is a copper-containing metalloglycoprotein that catalyzes several steps in the melanin pigment biosynthetic pathway; the hydroxylation of tyrosine to L-3,4-dihydroxyphenylalanine (dopa) and the subsequent oxidation of dopa to dopaquinone. It has been proposed that tyrosinase is also able to oxidize 5,6-dihydroxyindole (DHI), a later product in the melanogenic pathway, to indole-5,6-quinone. Tyrosinase enzymatic activity is deficient in patients with classic type I oculocutaneous albinism (OCA), and more than 50 distinct mutations have now been identified in the tyrosinase genes of such patients. To determine the effects of the various tyrosinase gene mutations on the catalytic activities of the enzyme, we carried out site-directed mutagenesis of human tyrosinase cDNA, transiently expressed the mutant cDNAs in transfected HeLa cells, and assayed the resultant encoded proteins for tyrosine hydroxylase, dopa, and DHI oxidase activities, and resulting melanin production. The tyrosine hydroxylase activity of normal tyrosinase is thermostable, whereas its dopa oxidase and DHI oxidase activities are temperature-sensitive. Although all amino acid substitutions tested generally affected the dopa oxidase and DHI oxidase activities in parallel, several exerted distinctly different effects on the tyrosine hydroxylase activities. Together, these results confirm the DHI oxidase activity of mammalian tyrosinase and suggest that the dopa oxidase and DHI oxidase activities of tyrosinase share a common catalytic site, whereas the tyrosine hydroxylase catalytic site is at least partially distinct in the tyrosinase polypeptide.

Electron spin resonance spectroscopy has been used to demonstrate production of semiquinone-free radicals from the oxidation of the catechol estrogens 2- and 4-hydroxyestradiol and 2,6- and 4,6-dihydroxyestradiol. Radicals were generated either enzymatically (using horseradish peroxidase-H2O2 or tyrosinase-O2) or by autoxidation, and were detected as their complexes with spin-stabilizing metal ions (Zn2+ and/or Mg2+). In the peroxidase system, radicals are produced by one-electron oxidation of the catechol estrogen and their decay is by a second-order pathway, consistent with their disproportionation to quinone and catechol products. With tyrosinase-O2, radical generation occurs indirectly. Initial hydroxylation of phenolic estrogen (at either the 2- or 4-position) gives a catechol estrogen in situ; subsequent two-electron oxidation of the catechol to the quinone, followed by reverse disproportionation, leads to the formation of radicals. A competing mechanism for radical production involves autoxidation of the catechol. Results obtained from the estrogen systems have been compared with those from the model compound 5,6,7,8-tetrahydro-2-naphthol.

Chloride ion (Cl-) effects on chloroperoxidase (CPO)-catalyzed peroxidation of catechol were used to probe the involvement of Cl- in CPO reactions. High concentrations of Cl- inhibit catechol peroxidation by competing with hydrogen peroxide (KI = 370 mM). However, at lower concentrations, Cl- is a linear competitive activator versus catechol (KDC = 35 mM). Addition of good halogenation substrates to the peroxidatic reaction mixture converts Cl- from a competitive activator to a competitive inhibitor. The KI (10 mM) for this halogenation substrate promoted Cl- inhibition is equivalent to the KM (11 mM) for Cl- in CPO-catalyzed halogenation reactions. During this inhibition, the halogenation substrate is consumed and, at the point where its consumption is complete, Cl- again becomes an activator. Also, at 2.0 mM hydrogen peroxide, CPOs chlorination reaction and its Cl- -activated peroxidatic reaction have similar apparent kcat values. All data are consistent with a mechanism in which Cl- competes with catechol for binding to CPO Compound I. Catechol binding initiates the Cl- -independent path, in which Compound I acts as the oxidizing agent for catechol. When Cl- binds to Compound I, it reacts to yield the enzymatic chlorinating intermediate which is responsible for either the oxidation of catechol in the Cl- -dependent path or the chlorination of substrates in the halogenation pathway. Cl- activation of the peroxidatic reaction is due to a shift from the Cl- -independent pathway to the Cl- -dependent process. The mechanism is unique in that exclusion of the substrate from its primary binding site leads to an increase in the catalytic efficiency of the reaction. This catechol-Cl- system also offers further potential for probing the specificity and chemistry of the key enzymatic intermediates in haloperoxidase-catalyzed reactions.

Tyrosinase, which usually catalyzes the conversion of o-diphenols to o-benzoquinones, catalyzed an unusual oxidative dimerization of 1,2-dehydro-N-acetyl-dopamine to a benzodioxan derivative. The identity of the product was confirmed by UV, IR spectra, and NMR studies. During the oxidation, generation of a transient reactive intermediate could be witnessed by its characteristic visible absorption spectrum. Typical phenoloxidase inhibitors such as phenylthiourea, potassium cyanide, sodium azide, and sodium fluoride drastically inhibited the above reaction. Mimosine, a known competitive inhibitor of o-diphenoloxidase activity, also inhibited the new reaction competitively, suggesting that both the observed oxidative dimerization and the conventional quinone production are catalyzed by the same active site copper of tyrosinase. Based on our earlier findings (Sugumaran, M., and Lipke, H. (1983) FEBS Lett. 155, 65-68; Sugumaran, M. (1986) Biochemistry 25, 4489-4492) that phenoloxidases can produce quinone methides from certain 4-alkylcatechols, possible mechanisms for this new reaction are presented.

gamma-L-Glutaminyl-[3,5-3H]4-hydroxybenzene was synthesized in order to study the kinetics of its hydroxylation by tyrosinase purified from Agaricus bisporus and to explore its role in the induction of the dormant state in the spores of this species. It was found to be unique among the monophenolic substrates for tyrosinase in that the lag period for the hydroxylation reaction decreased with increasing substrate concentration. Unlike previously studied compounds, this phenol appeared to function as an electron donor, allowing it to act as its own co-substrate in the hydroxylation reaction. Its catechol product, gamma-L-glutaminyl-3,4-dihydroxybenzene, was found to be a superior co-substrate, yielding its electrons more readily (oxidation peak potential +0.18 V as compared with +0.65 V for the phenol). In situ periodate oxidation of gamma-L-glutaminyl-3,4-dihydroxybenzene to gamma-L-glutaminyl-3,4-benzoquinone confirmed the co-substrate role of the catechol in the hydroxylation reaction. The tyrosinase-mediated oxidation of gamma-L-glutaminyl-3,4-dihydroxybenzene to gamma-L-glutaminyl-3,4-benzoquinone occurred with an apparent Km = 1.54 mM and Vmax = 0.36 mmol/min/mg of enzyme. gamma-L-Glutaminyl-4-hydroxybenzene acted as an inhibitor of the oxidation reaction.

An oxygenated tyrosinase (T'') is formed by addition of H2O2 to resting mushroom enzyme (Jolley, R. L., Jr., Evans, L. H. & Mason, H. S. (1971) The Second International Symposium on Oxidases and Redox Systems, Memphis, Tennessee, in press; (1972) Biochem. Biophys. Res. Commun. 46, 878). The reactivity of T'' toward monophenolic tyrosinase substrates was investigated by the stopped flow method. The first-order rate constants for T'' disappearance in the presence of p-hydroxybenzaldehyde and p-hydroxybenzoate were about 0.5 and 0.3 s-1, respectively. These rates were sufficiently large to support the over-all enzyme-catalyzed reactions and were not affected by H2O2 concentrations within the concentration ranges examined. T'' appears to be the first oxygenated intermediate detected with a copper oxidase.

Tyrosinase (o-diphenol:O2 oxidoreductase, EC 1.10.3.1) has been purified from a commercial lyophilized powder prepared from edible mushrooms. Specific activities toward dl-dihydroxyphenylalanine and catechol, homogeneity, molecular weight, absorption spectrum, amino acid composition, and copper content of the preparation are comparable to other purified samples of tyrosinase prepared from fresh mushrooms. Circular dichroism of the enzyme reveals an unusually shaped envelope of negative ellipticity bands in the deep ultraviolet (196 to 250 mµ), which is difficult to explain by a combination of the usual peptide conformational forms. Small positive ellipticity bands ([θ] per 32,000 mol wt ≅ 30,000) are associated with the near ultraviolet absorption of the aromatic amino acid side chains. Kinetic constants for the oxidation of a homologous series of catechol substrates substituted in position 4 by groups —H, —SCN, —COCH3, —CHO, —CN, and —NO2 have been determined. As the electron-withdrawing ability of the substituent increases, Km and kcat decrease in the order H g SCN g COCH3 g CHO g CN g NO2. Except for the kcat values for the two most slowly oxidized substrates, both Km and kcat conform to Hammett relationships when the substituent parameter σ- is used. The ρ values are -1.01 ± 0.09 for Km and -2.49 ± 0.11 for kcat. The oxidation of pyrocatechol, 4-COCH3, and 4-CHO-catechol give a satisfactory isokinetic relationship between ΔH‡ and ΔS‡. These findings suggest that these substrates are oxidized by the same basic mechanism. Studies of the dependence of catechol oxidation on the concentration of oxygen show that Km for oxygen varies with the nature of the catechol substrate. With the exception of the pyrocatechol complex, the enzyme-substrate complexes for this series of catechols are saturated in air (20.95% O2). The oxygen kinetics suggest a sequential mechanism for the binding of catechol and oxygen. Detailed studies of the pH dependence of Km and kcat show the presence of a group in the enzyme whose pKa changes upon the binding of a catechol molecule. Inhibition of tyrosinase by benzoic acid and cyanide show that the former is competitive with catechol and noncompetitive with oxygen, while the latter is competitive with oxygen and noncompetitive with catechol. These results are taken to indicate two distinct substrate binding sites on the enzyme, one with a high affinity for aromatic compounds including phenolic substrates, the other for metal-binding agents and oxygen. The latter site presumably involves enzyme copper. Binding studies with 14C-benzoic acid suggest that there are two binding sites for benzoic acid per mole of tetramer (mol wt = 128,000). The equilibrium constant for this binding is the same as the Ki measured for competitive inhibition of catechol oxidation by benzoic acid. Catechols are activators of the phenolase reaction of tyrosinase, and tyrosine is shown to be an inhibitor of this activation of its own oxidation in a manner suggesting that there is a third binding site on the enzyme for an "activator" catechol or phenol.

Two new intermediates were identified in the protocatechuate pathway of Pseudomonas putida. The first of these, γ-carboxymuconolactone (γ-carboxy-γ-carboxymethyl-Δα-butenolide), is the product of the enzymic lactonization of β-carboxy-cis, cis-muconate. Enzymic decarboxylation of γ-carboxymuconolactone gives rise to β-ketoadipate enollactone (γ-carboxymethyl-Δβ-butenolide), the second newly discovered intermediate in the protocatechuate pathway. β-Ketoadipate enol-lactone, which was isolated and physically characterized, is also an intermediate in the catechol pathway; the catechol and protocatechuate pathways converge at this point. β-Ketoadipate enol-lactone is hydrolyzed to β-ketoadipate by an enzyme which is essential for utilization of either catechol or protocatechuate. Studies with Moraxella lwoffii showed that this organism also degrades protocatechuate and catechol by the pathways characteristic of P. putida. Elucidation of the bacterial pathway for the dissimilation of protocatechuate revealed that the three step-reactions responsible for the conversion of this compound to β-ketoadipate enol-lactone are analogous with the step-reactions responsible for the conversion of catechol to β-ketoadipate enol-lactone.

3,4-Dihydroxy-l-phenylalanine (dopa) has been shown to account for the cofactor activity for tyrosinase contained in extracts of hamster melanoma. Dopa has been identified by several different comparisons with an authentic sample and by conversion to two derivatives. The amount of dopa in the tumor (4 to 8 µg per g) has been estimated by cofactor assay and fluorometry. A Km value for dopa as a cofactor was estimated to be about 2 x 10-6 m, much lower than its Km as a sole substrate (5 x 10-4 m).

Study of the regulation of the syntheses of enzymes of the catechol and protocatechuate pathways in Pseudomonas putida has shown that two groups of enzymes are subject to coordinate control. cis,cis-Muconate-lactonizing enzyme and muconolactone isomerase, which are uniquely associated with the catechol pathway, constitute the first coordinate block of enzymes. The synthesis of these enzymes, as well as that of catechol oxygenase (which is regulated independently), seems to be induced by cis,cis-muconate. The second coordinate block of enzymes comprises β-carboxy-cis,cis-muconate-lactonizing enzyme and γ-carboxymuconolactone decarboxylase, which are uniquely associated with the protocatechuate pathway, and β-ketoadipate enol-lactone hydrolase, which is functional in both the protocatechuate and the catechol pathways. This group of enzymes seems to be induced by β-ketoadipate or β-ketoadipyl coenzyme A. Moraxella lwoffii, which degrades protocatechuate and catechol through identical step-reactions, regulates the synthesis of the enzymes mediating these conversions by a different mechanism.