Apparent Enzyme Activity Research Articles

The role of human leukocyte antigen E and G in HIV infection

The role of human leukocyte antigen E and G in HIV infection

Development of Crack-Free Alumina Sol-gel/Poly(vinyl Alcohol) Membranes for Glucose Oxidase Immobilization

A simple procedure to incorporate PVA into alumina sol-gel membrane was investigated as a suitable support material for glucose oxidase. The alumina sol was prepared using aluminum iso-propoxide via the sol-gel process. PVA was employed as the organic binder to enhance the mechanical strength of the fragile sol-gel membranes. The ability of the hybrid membrane to retain glucose oxidase and the apparent enzyme activities were studied. The resulting composite membranes were found to be crack-free, stable, and still very active after 60 days. However, the enzyme leakage period was observed to be quite long. The enzyme was still leaking from the membrane after more than 10 days albeit at a very low level.

Effects of diets with distinct protein-to-carbohydrate ratios on nutrient digestibility, growth performance, body composition and liver intermediary enzyme activities in gilthead sea bream ( Sparus aurata, L.) fingerlings

The effect of replacing dietary protein with gelatinized cornstarch (GCS) on apparent digestibility coefficient (ADC), body composition, growth performance and liver enzyme activities involved in control of intermediary metabolism, was studied in Sparus aurata L. Fingerlings of S. aurata were fed 93 days three diets containing 63% protein and 5% gelatinized cornstarch (LC diet), 54% protein and 18% GCS (MC diet) or 47% protein and 26% GCS (HC diet). Diet HC gave ADC values for carbon, nitrogen and dry matter that were significantly below the corresponding values of the other diets. Fish on MC diet registered higher fresh weight than fish on LC and HC, and higher specific growth rate (SGR) than fish on HC. The lipid body content ranked in the order HC > MC > LC. High correlations between carbohydrate level and liver enzyme activity were found for pyruvate kinase, glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and alanine aminotransferase. For cultures of S. aurata, we conclude that carbohydrates like GCS could replace dietary protein, enhance growth rate and reduce nitrogen losses to the ambient waters when used at levels below 20%.

Cholesterol biosensor based on N-(2-aminoethyl)-3-aminopropyl-trimethoxysilane self-assembled monolayer

Cholesterol oxidase (ChOx) has been covalently immobilized onto two-dimensional self-assembled monolayer (SAM) of N-(2-aminoethyl)-3-aminopropyl-trimethoxysilane (AEAPTS) deposited on the indium–tin oxide (ITO) coated glass plates using N-ethyl- N′-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide (EDC/NHS) chemistry. These ChOx/AEAPTS/ITO bioelectrodes are characterized using contact angle (CA) measurements, UV–visible spectroscopy, atomic force microscopy (AFM), electrochemical impedance technique, and Fourier transform infrared (FT–IR) technique. The covalently immobilized ChOx-modified AEAPTS bioelectrodes are used for the estimation of cholesterol in solution using UV–visible technique. These cholesterol sensing bioelectrodes show linearity as 50 to 500 mg/dl for cholesterol solution, detection limit as 25 mg/dl, sensitivity as 4.499 × 10 −5 Abs (mg/dl) −1, K m value as 58.137 mg/dl (1.5 mM), apparent enzyme activity as 1.81 × 10 −3 U cm −2, shelf life of approximately 10 weeks, and electrode reusability as 10 times.

Effect of varying dietary protein levels on growth and production of Chanos chanos (Forsskal) in inland saline groundwater: laboratory and field studies

Two experiments were conducted to investigate the effects of varying dietary protein (35–45%) and energy levels (17.34–19.44 kJ g−1) on the growth performance of milkfish (Chanos chanos) maintained under laboratory (experiment 1) and field conditions (Experiment 2) in inland saline groundwater. The results of experiment 1 (initial weight of fish: 0.25 g) revealed that, irrespective of the protein source (fish meal or processed full fat soybean), fish fed a diet containing 40% protein showed significantly (p<0.05) higher growth in terms of live weight gain and specific growth rate, low feed conversion ratio [1.71 (fish meal) and 1.58 (soybean)], high nutrient retention (gross protein retention: 28.59 and 31.05%; gross energy retention: 24.23 and 26.04%), apparent protein digestibility (81.74 and 85.91%) and digestive enzyme activity (specific protease and amylase). An observation on the postprandial excretion of metabolites (N–NH4 and o–PO4) indicated significantly (p<0.05) low levels of these metabolites in aquaria where the fish were fed 40% dietary protein irrespective of the protein source. The results of experiment 2 also indicate (initial weight of fish: 4.43 g) a significantly (p<0.05) higher growth performance (weight gain, biomass, specific growth rate, growth day−1 and fish production) in ponds where the fish were fed 40% dietary protein. Irrespective of the experimental conditions (laboratory/field), feeding the fish higher dietary protein levels (beyond 40%) not only repressed growth performance but also affected proximate composition by lowering protein accumulation and energy assimilation. An investigation on the effects of feeds on water quality parameters revealed that values for total alkalinity, NH4–N, NO3–N, turbidity, total dissolved solids and parameters indicative of productivity (chlorophyll a, net primary productivity and plankton population) increased significantly (p<0.05) from treatments 1–3 (35–40% protein) and declined thereafter, in treatment 4 (42% dietary protein), indicating that water quality characteristics correlated well with fish growth. The results suggest that in order to obtain a high-yield in milkfish culture system the fish should be fed supplementary diets containing appropriate (40%) protein levels, especially when the stocking rates are high.

Phospholipase D1 Regulates Cell Migration in a Lipase Activity-independent Manner

Cell migration, a complex biological process, requires dynamic cytoskeletal remodeling. Phospholipase D (PLD) generates phosphatidic acid, a lipid second messenger. Although PLD activity has been proposed to play a role in cytoskeletal rearrangement, the manner in which PLD participates in the rearrangement process remains obscure. In this study, by silencing endogenous PLD isozymes using small interfering RNA in HeLa cells, we demonstrate that endogenous PLD1 is required for the normal organization of the actin cytoskeleton, and, more importantly, for cell motility. PLD1 silencing in HeLa cells resulted in dramatic changes in cellular morphology, including the accumulation of stress fibers, as well as cell elongation and flattening, which appeared to be caused by an increased number of focal adhesions, which ultimately culminated in enhanced cell-substratum interactions. Accordingly, serum-induced cell migration was profoundly inhibited by PLD1-silencing. Moreover, the augmented cell substratum interaction and retarded cell migration induced by PLD1-silencing could be restored by the adding back not only of wild type, but also of lipase-inactive PLD1 into knockdown cells. Taken together, our results strongly suggest that endogenous PLD1 is a critical factor in the organization of the actin-based cytoskeleton, with regard to cell adhesion and migration. These effects of PLD1 appear to operate in a lipase activity-independent manner. We also discuss the regulation of Src family kinases by PLD1, as related to the modulation of Pyk2 and cell migration.

Kinetic Studies of Site-Directed Mutational Isomalto-dextranase-Catalyzed Hydrolytic Reactions on a 27 MHz Quartz-Crystal Microbalance

A quartz-crystal microbalance (QCM) technique was applied to analyze effects of site-directed mutagenesis of a glycosidase (isomalto-dextranase) on the hydrolysis mechanism of the substrate binding (k(on), k(off), and K(d)) and the catalytic process (k(cat)), separately, by using a dextran-immobilized QCM in buffer solution. D266N, D198N, and D313N mutants, which are predicted as critical residues of the isomalto-dextranase hydrolytic activity, dramatically decreased the apparent enzyme activity. The D266N mutant, however, did not change the substrate binding ability (K(d)), and the D198N and D313N mutants largely increased K(d) values due to the increase of k(off) and/or the decrease of k(on) values, as well as the negatively small k(cat) values. From these results, we estimate the reaction mechanism, in which Asp266 acts as only a general acid in the catalytic process, Asp198 acts as both nucleophile in the catalytic process and binding the substrate, and Asp313 acts as only the substrate binding.

N-linked glycosylation of native and recombinant cauliflower xyloglucan endotransglycosylase 16A.

The gene encoding a XET (xyloglucan endotransglycosylase) from cauliflower ( Brassica oleracea var. botrytis ) florets has been cloned and sequenced. Sequence analysis indicated a high degree of similarity to other XET enzymes belonging to glycosyl hydrolase family 16 (GH16). In addition to the conserved GH16 catalytic sequence motif EIDFE, there exists one potential N-linked glycosylation site, which is also highly conserved in XET enzymes from this family. Purification of the corresponding protein from extracts of cauliflower florets allowed the fractionation of a single, pure glycoform, which was analysed by MS techniques. Accurate protein mass determination following the enzymic deglycosylation of this glycoform indicated the presence of a high-mannose-type glycan of the general structure GlcNAc2Man6. LC/MS and MS/MS (tandem MS) analysis provided supporting evidence for this structure and confirmed that the glycosylation site (underlined) was situated close to the predicted catalytic residues in the conserved sequence YLSSTNNEHDEIDFEFLGNRTGQPVILQTNVFTGGK. Heterologous expression in Pichia pastoris produced a range of protein glycoforms, which were, on average, more highly mannosylated than the purified native enzyme. This difference in glycosylation did not influence the apparent enzymic activity of the enzyme significantly. However, the removal of high-mannose glycosylation in recombinant cauliflower XET by endoglycosidase H, quantified by electrospray-ionization MS, caused a 40% decrease in the transglycosylation activity of the enzyme. No hydrolytic activity was detected in native or heterologously expressed BobXET16A, even when almost completely deglycosylated.

Calcium alginate entrapment of the yeast Rhodosporidium toruloides for the kinetic resolution of 1,2-epoxyoctane.

Resting cells of the yeast Rhodosporidium toruloides (UOFS Y-0471) were immobilised in calcium alginate beads for the enantioselective kinetic resolution of racemic-1,2-epoxyoctane. The initial activity exhibited by immobilised cells was almost 50% lower than that of the free counterpart but was extremely stable when compared to the free cells. The concentration of the immobilised biomass had no effect on apparent enzyme activity but did lead to a decrease in single cell activity. An increase in both the alginate and CaCl2 concentrations used for bead preparation led to a decrease in enzyme stability. An increase in the alginate concentration led to an increase in bead diameter. The stoichiometric equation for cross-linking of alginate was only obeyed when CaCl2 concentrations higher than 0.4 M were utilised for bead preparation.

Structural Determinants of Aromatase Cytochrome P450 Inhibition in Substrate Recognition Site-1

The porcine gonadal form of aromatase cytochrome P450 (P450arom) exhibits higher sensitivity to inhibition by the imidazole, etomidate, than the placental isozyme. The residue(s) responsible for this functional difference was mapped using chimeragenesis and point mutation analysis of the placental isozyme, and the kinetic analysis was conducted on native and mutant enzymes after overexpression in insect cells. The etomidate sensitivity of the placental isozyme was markedly increased by substitution of the predicted substrate recognition site-1 (SRS-1) and essentially reproduced that of the gonadal isozyme by substitution of SRS-1 and the predicted B helix. A single isoleucine (I) to methionine (M) substitution at position 133 of the placental isozyme (I133M) was proven to be the critical residue within SRS-1. Residue 133 is located in the B′-C loop and has been shown to be equally important in other steroid-metabolizing P450s. Single point mutations (including residues 110, 114, 120, 128, 137, and combinations thereof among others) and mutation of the entire B and C helixes were without marked effect on etomidate inhibitory sensitivity. The same mutation (I133M) introduced into human P450arom also markedly increased etomidate sensitivity. Mutation of Ile133 to either alanine (I133A) or tyrosine (I133Y) decreased apparent enzyme activity, but the I133A mutant was sensitive to etomidate inhibition, suggesting that it is Ile133 that decreases etomidate binding rather than Met133 increasing enzyme sensitivity. Androstenedione turnover and affinity were similar for the I133M mutant and the native placental isozyme. These data suggest that Ile133 is a contact residue in SRS-1 of P450arom, emphasize the functional conservation that exists in SRS-1 of a number of steroid-hydroxylating P450 enzymes, and suggest that substrate and inhibitor binding are dependent on different contact points to varying degrees.

Structural determinants of aromatase cytochrome p450 inhibition in substrate recognition site-1.

The porcine gonadal form of aromatase cytochrome P450 (P450arom) exhibits higher sensitivity to inhibition by the imidazole, etomidate, than the placental isozyme. The residue(s) responsible for this functional difference was mapped using chimeragenesis and point mutation analysis of the placental isozyme, and the kinetic analysis was conducted on native and mutant enzymes after overexpression in insect cells. The etomidate sensitivity of the placental isozyme was markedly increased by substitution of the predicted substrate recognition site-1 (SRS-1) and essentially reproduced that of the gonadal isozyme by substitution of SRS-1 and the predicted B helix. A single isoleucine (I) to methionine (M) substitution at position 133 of the placental isozyme (I(133)M) was proven to be the critical residue within SRS-1. Residue 133 is located in the B'-C loop and has been shown to be equally important in other steroid-metabolizing P450s. Single point mutations (including residues 110, 114, 120, 128, 137, and combinations thereof among others) and mutation of the entire B and C helixes were without marked effect on etomidate inhibitory sensitivity. The same mutation (I(133)M) introduced into human P450arom also markedly increased etomidate sensitivity. Mutation of Ile(133) to either alanine (I(133)A) or tyrosine (I(133)Y) decreased apparent enzyme activity, but the I(133)A mutant was sensitive to etomidate inhibition, suggesting that it is Ile(133) that decreases etomidate binding rather than Met(133) increasing enzyme sensitivity. Androstenedione turnover and affinity were similar for the I(133)M mutant and the native placental isozyme. These data suggest that Ile(133) is a contact residue in SRS-1 of P450arom, emphasize the functional conservation that exists in SRS-1 of a number of steroid-hydroxylating P450 enzymes, and suggest that substrate and inhibitor binding are dependent on different contact points to varying degrees.

Enzyme-carrying polymeric nanofibers prepared via electrospinning for use as unique biocatalysts.

Improvement of catalytic efficiency of immobilized enzymes via materials engineering was demonstrated through the preparation of bioactive nanofibers. Bioactive polystyrene (PS) nanofibers with a typical diameter of 120 nm were prepared and examined for catalytic efficiency for biotransformations. The nanofibers were produced by electrospinning functionalized PS, followed by the chemical attachment of a model enzyme, alpha-chymotrypsin. The observed enzyme loading as determined by active site titration was up to 1.4% (wt/wt), corresponding to over 27.4% monolayer coverage of the external surface of nanofibers. The apparent hydrolytic activity of the nanofibrous enzyme in aqueous solutions was over 65% of that of the native enzyme, indicating a high catalytic efficiency as compared to other forms of immobilized enzymes. Furthermore, nanofibrous alpha-chymotrypsin exhibited a much-improved nonaqueous activity that was over 3 orders of magnitude higher than that of its native counterpart suspended in organic solvents including hexane and isooctane. It appeared that the covalent binding also improved the enzyme's stability against structural denaturation, such that the half-life of the nanofibrous enzyme in methanol was 18-fold longer than that of the native enzyme.

Lack of significant differences in the corrected activity of lysophospholipase D, producer of phospholipid mediator lysophosphatidic acid, in incubated serum from women with and without ovarian tumors.

Several studies have shown that lysophosphatidic acid (LPA), a phospholipidic chemical mediator, is relevant to the pathogenesis of ovarian carcinoma. Higher plasma levels of LPA have been reported in patients with ovarian carcinoma than in healthy patients, and LPA is known to activate ovarian carcinoma cells. To determine the reason for the increased plasma LPA levels in ovarian carcinoma patients, we compared the activities of serum lysophospholipase D, a novel LPA-producing metallo-enzyme, in healthy volunteers, patients with benign ovarian tumor, and patients with ovarian carcinoma. Lysophospholipase D activity was assessed by measuring the percentage conversion of [14C]palmitoyl-lysophosphatidylcholine (LPC) added to human serum. The apparent enzyme activities were corrected based on the serum levels of palmitoyl-LPC determined by gas-liquid chromatography after its purification and conversion to fatty acid methyl esters. The apparent activity of lysophospholipase D in serum preparations from four patients with ovarian carcinoma at Stage IV was significantly higher than those from five healthy subjects, five patients with benign ovarian tumors, and fourteen patients with ovarian carcinoma at Stages I (n = 5), II (n = 4), and III (n = 5). The serum levels of LPC, an endogenous substrate of lysophospholipase D, in ovarian carcinoma patients were less than those in patients with benign ovarian tumors. There were no significant differences in the corrected lysophospholipase D activity for the LPC levels in healthy women, patients with benign ovarian tumors, and patients with ovarian carcinoma at various stages. The current results suggest that lysophospholipase D is not associated with the elevated plasma levels of LPA in ovarian carcinoma patients previously reported, although only a limited number of patients were analyzed.

Pronounced activity of enzymes through the incorporation into the core of polyion complex micelles made from charged block copolymers

Compartmentalization of enzymes in the nanometric-scaled container, purposing to improve their stability and availability, has recently attracted a strong interest in the field of pharmaceutics. In this study, the enzymatic activity of lysozyme in the core of polyion complex (PIC) micelles, which were formed from egg white lysozyme and poly(ethylene glycol)–poly(α,β-aspartic acid) block copolymer (PEG-P(Asp)), was evaluated using a colorimetric method. Apparent enzymatic activity of lysozyme entrapped in the core of PIC micelles remarkably increased compared to that of free lysozyme, which is mainly attributed to a decrease in the observed Michaelis constant ( K m,obs). The reciprocal of the K m,obs values nicely correlated to the corona thickness of PIC micelles, suggesting that the corona layer of PIC micelle may act as the reservoir of the substrate, p-nitrophenyl penta- N-acetyl-β-chitopentaoside. This result indicates that the enzymatic activity can be controlled by changing the corona thickness of PIC micelles through a variation in the mixing ratio of PEG-P(Asp) to lysozyme. This type of PIC micelle system entrapping enzyme in the core might be useful for the design of diagnostic as well as targetable therapeutic systems of enzyme including antibody-directed enzyme prodrug therapy (ADEPT).

Performance of pigs post-weaning fed cereal-based diets with an enzyme complex added either before or after pelleting

The responses of pigs to diets supplemented with enzymes have been variable (review by Partridge, 2000). The situation has not been eased due to an uncertainty over the usefulness of laboratory analyses of enzyme activity as a measure of performance. One issue has been the temperature of feed pelleting in which laboratory assays for apparent enzyme activity have shown very low levels compared with the known level included in the feed, whereas experimental data have indicated typical poultry responses from the enzyme-supplemented feed (Bedford and Pack, 1998). The experiment reported here studied the performance of pigs fed diets to which enzymes were added either pre-or post- pelleting (which was conducted at 850 C).

Assay of microsomal oxysterol 7α-hydroxylase activity in the hamster liver by a sensitive method: in vitro modulation by oxysterols

A method of assaying hepatic cytochrome P-450, oxysterol 7α-hydroxylase (CYP7B), was developed by combining the use of 25-[26,27- 3H]hydroxycholesterol as a substrate and hydroxypropyl-β-cyclodextrin as a substrate vehicle. When these assay conditions were tested, an undesirable transformation was observed of the reaction product, 7α,25-dihydroxycholesterol, into 3-oxo-7α,25-dihydroxy-4-cholesten by the activity of 3β-hydroxy-Δ 5-C 27 steroid oxydoreductase, a microsomal NAD + and NADP + dependent enzyme of bile acid metabolism. A great improvement was reached by using a continuous NADPH generating system which constantly re-transforms NADP + into NADPH, thus inhibiting this activity. This improved CYP7B assay, comparable to our previously described assay for cholesterol 7α-hydroxylase (CYP7A), allowed a 3-fold increase of the apparent enzyme activity. The possibility to simultaneously measure CYP7A and CYP7B activities on the same microsomal preparation was investigated. A marked decrease (−33%) in the CYP7B activity was noticed, while that of CYP7A remained unchanged. The CYP7B activity was observed to be inhibited by cholesterol (−30%) and also by the oxysterols 7α-hydroxycholesterol (−21%), 7β-hydroxycholesterol (−25%) and epicoprostanol (−20%), and by cyclosporin A (−26%). It can be concluded that this sensible and easy to perform CYP7B assay allows to observe, at least in vitro, a modulation of the enzyme activity by oxysterols.

Widespread expression of tartrate-resistant acid phosphatase (Acp 5) in the mouse embryo.

Tartrate-resistant acid phosphatase (TRAP, Acp 5) is considered to be a marker of the osteoclast and studies using 'knockout' mice have demonstrated that TRAP is critical for normal development of the skeleton. To investigate the distribution of TRAP in the mammalian embryo, cryostat sections of 18 d murine fetuses were examined by in situ hybridisation, immunohistochemistry and histochemical reactions in situ. Abundant expression of TRAP mRNA was observed in the skin and epithelial surfaces of the tongue, oropharynx and gastrointestinal tract including the colon, as well as the thymus, ossifying skeleton and dental papillae. TRAP protein was identified at the same sites, but the level of expression in the different tissues did not always correlate with apparent enzyme activity. The findings indicate that abundant TRAP expression is not confined to osteoclasts in bone, but occurs in diverse tissues harbouring cells of bone marrow origin, including dendritic cells and other cells belonging to the osteoclast/macrophage lineage.

Sugar Surfactant-Based Solutions as Host Systems for Enzyme Activity Measurements

We used fluorogenic substrates, namely, alkyldiacyl glycerols labeled with a fluorophore and a fluorescence quencher, to measure lipase activities. For this optical lipase assay it is necessary that the water-insoluble fluorogenic substrates are dispersed in an aqueous medium in the presence of appropriate detergents in order to obtain well-defined and reproducible systems with high optical transparency. The activity of lipases critically depends on the supramolecular organization of the substrate. Therefore, we tested different solutions containing nonionic surfactants and organic phases for their potential use as host systems for fluorogenic lipids. We then determined the pseudoternary phase diagram for the system buffer−alkyl polyglucoside−hexanol, which showed the highest apparent enzyme activities, in the buffer corner. We performed structural investigations on micellar sugar surfactant solutions within the L1 phase with and without hexanol using small-angle neutron scattering (SANS), small-angle X-ray scattering (SAXS), and dynamic light scattering (DLS) in order to investigate a possible correlation between lipase activity and the structure of the aggregates present in the host system.

TEMPERATURE-SENSITIVE LATEX PARTICLES FOR IMMOBILIZATION OF α-AMYLASE

ABSTRACT Poly(methyl methacrylate-co-N-isopropylacrylamide-co-N-acryloxysuccin-imide) (poly(MMA/NIPAAni/NAS)) latex particles were prepared by emulsifier-free emulsion polymerization. The latex particles could reversibly flocculate and disperse when the solution temperature was kept above and below the characteristic lower critical solution temperature (LCST). The LCST of the latex dispersion could be adjusted lower or higher by increasing or decreasing, respectively, the ionic strength of the solution.α-Amylase was immobilized onto the latex particles by reacting with the succinimide groups on NAS. It was demonstrated that the ct-amylase immobilized latex particles could be separated by thermoflocculation. After 5 repeated thermocycles, 83 % of the latex particles could be recovered, and the apparent enzymatic activity could be retained as high as 78percnt;.

Myrothecium verrucaria bilirubin oxidase and its mutants for potential copper ligands.

Bilirubin oxidase (EC:1.3.3.5) purified from a culture medium of Myrothecium verrucaria MT-1 (authentic enzyme) catalyzes the oxidation of bilirubin to biliverdin in vitro and recombinant enzyme (wild type) was obtained by using an overexpression system of the bilirubin oxidase gene with Aspergillus oryzae harboring an expression vector. The absorption and ESR spectra showed that both bilirubin oxidases are multicopper oxidases containing type 1, type 2, and type 3 coppers similar to laccase, ascorbate oxidase, and ceruloplasmin. Site-directed mutagenesis has been performed for the possible ligands of each type of copper. In some mutants, Cys457 --> Val, Ala, His94 --> Val, and His134.136 --> Val, type 1 and type 2 copper centers were perturbed completely and the enzyme activity was completely lost. Differing from the holoenzyme, these mutants showed type 3 copper signals. However, the optical and magnetic properties characteristic of type 1 copper were retained even by mutating one of the type 1 copper ligands, i.e., a mutant, Met467 --> Gly, showed a weak but apparent enzyme activity. A double mutant His456.458 --> Val had only type 1 Cu, showing a blue band at 600 nm (epsilon = 1.6 x 10(3)) and an ESR signal with very narrow hyperfine splitting (A parallel = 7.2 x 10(-)3 cm-1). Since the type 2 and type 3 coppers are not present, the mutant did not show enzyme activity. These results strongly imply that the peculiar sequence in bilirubin oxidase, His456-Cys457-His458, forms an intramolecular electron-transfer pathway between the type 1 copper site and the trinuclear center composed of the type 2 and type 3 copper sites.