Ternary copper complexes and manganese (III) tetrakis(4-benzoic acid) porphyrin catalyze peroxynitrite-dependent nitration of aromatics

Peroxynitrite is a powerful oxidant formed in biological systems from the reaction of nitrogen monoxide and superoxide and is capable of nitrating phenols at neutral pH and ambient temperature. This peroxynitrite-mediated nitration is catalyzed by a number of Lewis acids, including CO2 and transition-metal ion complexes. Here we studied the effect of ternary copper-(II) complexes constituted by a 1,10-phenanthroline and an amino acid as ligands. All the complexes studied accelerate both the decomposition of peroxynitrite and its nitration of 4-hydroxyphenylacetic acid at pH > 7. The rate of these reactions depends on the copper complex concentration in a hyperbolic plus linear manner. The yield of nitrated products increases up to 2.6-fold with respect to proton-catalyzed nitration and has a dependency on the concentration of copper complexes which follows the same function as observed for the rate constants. The manganese porphyrin complex, Mn(III)tetrakis(4-benzoic acid)porphyrin [Mn(tbap)], also promoted peroxynitrite-mediated nitration with an even higher yield (4-fold increase) than the ternary copper complexes. At pH = 7.5 +/- 0.2 the catalytic behavior of the copper complexes can be linearly correlated with the pKa of the phenanthroline present as a ligand, implying that a peroxynitrite anion is coordinated to the copper ion prior to the nitration reaction. These observations may prove valuable to understand the biological effects of these transition-metal complexes (i.e., copper and manganese) that can mimic superoxide dismutase activity and, in the case of the ternary copper complexes, show antineoplastic activity.     

Enhancement of radiation-inducible hepatic glutathione-S-transferases Ya, Yb1, Yb2, Yc1, and Yc2 gene expression by oltipraz: possible role in radioprotection

Variously modified metalloporphyrins offer a promising route to stable and active mimics of superoxide dismutase (SOD). Here we explore bromination on the pyrroles as a means of increasing the redox potentials and the catalytic activities of the copper and manganese complexes of a cationic porphyrin. Mn(II) and Cu(II) octabrominated 5,10,15,20-tetrakis-(N-methylpyridinium-4-yl) porphyrin, Mn(II)OBTMPyP4+, and Cu(II)OBTMPyP4+ were prepared and characterized. The rate constants for the porphyrin-catalyzed dismutation of O2.- as determined from the inhibition of the cytochrome c reduction are k(cat) = 2.2 x 10(8) and 2.9 x 10(6) M(-1) s(-1), i.e., IC50 was calculated to be 12 nM and 0.88 microM, respectively. The metal-centered half-wave potential was E(1/2) = +0.48 V vs NHE for the manganese compound. Cu(II)OBTMPyP4+ proved to be extremely stable, while its Mn(II) analog has a moderate stability, log K = 8.08. Nevertheless, slow manganese dissociation from Mn(II)OBTMPyP4+ enabled the complex to persist and exhibit catalytic activity even at the nanomolar concentration level and at biological pH. The corresponding Mn(III)OBTMPyP5+ complex exhibited significantly increased stability, i.e., demetallation was not detected in the presence of a 400-fold molar excess of EDTA at micromolar porphyrin concentration and at pH 7.8. The beta-substituted manganese porphyrin facilitated the growth of a SOD-deficient strain of Escherichia coli when present at 0.05 microM but was toxic at 1.0 microM. The synthetic approach used in the case of manganese and copper compounds offers numerous possibilities whereby the interplay of the type and of the number of beta substituents on the porphyrin ring would hopefully lead to porphyrin compounds of increased stability, catalytic activity, and decreased toxicity.     

The roles of His-64, Tyr-103, Tyr-146, and Tyr-151 in the epoxidation of styrene and beta-methylstyrene by recombinant sperm whale myoglobin

Previous studies demonstrated that at least two mechanisms are involved in the epoxidation of styrene and stilbene by myoglobin and H2O2 (Ortiz de Montellano, P. R., and Catalano, C. E. (1985) J. Biol. Chem. 260, 9265-9271). One mechanism, reaction of the olefin with the ferryl oxygen, preserves the olefin stereochemistry and incorporates an oxygen from H2O2 into the epoxide. The second mechanism, proposed to be a protein-mediated co-oxidation process, results in loss of stereochemistry and incorporation of an atom of molecular oxygen. To examine the role of individual residues in olefin epoxidation, we have examined the catalytic activities of the possible Tyr-->Phe mutants, the His-64-->Val mutant, and a protein combining all the tyrosine and histidine mutations. The latter protein is less stable than the other mutants and is the only one for which a protein radical is not detected in the reaction with H2O2. The Km and Vmax for styrene epoxidation range, respectively, from 0.3-8 mM and 12-35 pmol/min/nmol of protein. Incubation with H(2)18O2 results in 20-30% incorporation of labeled oxygen into the epoxide with all the mutants except Y103F/Y151F, Y146F/Y151F, H64V, and H64V/Y103F/Y146F/Y151F, for which 52, 58, 89, and 96% of the epoxide oxygen, respectively, is labeled. Oxidation of cis-beta-methylstyrene by wild-type myoglobin yielded a 54:46 ratio of cis- and trans-beta-methylstyrene oxides. The cis-isomer accounts for 47-100% of the epoxide produced by the mutant hemoproteins, with the two H64V mutants yielding almost exclusively the cis-epoxide. The oxygen in the cis-epoxide derives primarily or exclusively from H2O2 and that in the trans-epoxide from an alternative source. These results indicate that tyrosine residues may participate in, but are not essential for, protein-mediated epoxidation. In contrast, His-64 appears to be essential for co-oxidative epoxidation because in its absence olefin epoxidation is mediated almost exclusively by ferryl oxygen transfer.     

Epoxidation of olefins by cytochrome P450: evidence from site-specific mutagenesis for hydroperoxo-iron as an electrophilic oxidant

P450 cytochromes (P450) catalyze many types of oxidative reactions, including the conversion of olefinic substrates to epoxides by oxygen insertion. In some instances epoxidation leads to the formation of products of physiological importance from naturally occurring substrates, such as arachidonic acid, and to the toxicity, carcinogenicity, or teratogenicity of foreign compounds, including drugs. In the present mechanistic study, the rates of oxidation of model olefins were determined with N-terminal-truncated P450s 2B4 and 2E1 and their respective mutants in which the threonine believed to facilitate proton delivery to the active site was replaced by alanine. Styrene epoxidation, cyclohexene epoxidation and hydroxylation to give 1-cyclohexene-3-ol, and cis- or trans-butene epoxidation (without isomerization) and hydroxylation to give 2-butene-1-ol were all significantly decreased by the 2B4 T302A mutation. Reduced proton delivery in this mutant is believed to interfere with the activation of dioxygen to the oxenoid species, as shown earlier by decreased hydroxylation of several substrates and enhanced aldehyde deformylation via a presumed peroxo intermediate. Of particular interest, however, the T303A mutation of P450 2E1 resulted in enhanced epoxidation of all of the model olefins along with decreased allylic hydroxylation of cyclohexene and butene. These results and a comparison of the ratios of the rates of epoxidation and hydroxylation support the concept that two different species with electrophilic properties, hydroperoxo-iron (FeO2H)3+ and oxenoid-iron (FeO)3+, can effect olefin epoxidation. The ability of cytochrome P450 to use several different active oxidants generated from molecular oxygen may help account for the broad reaction specificity and variety of products formed by this versatile catalyst.     

Zirconia-Supported Copper Oxide—A Catalyst for Removal of Vehicular Exhaust Gas Pollutants

Copper oxide and zirconia-supported copper oxide catalysts were tested for their catalytic activity for carbon monoxide and propylene oxidation reactions. The synthesized catalyst was supported by wet impregnation on zirconia. The higher activity of zirconia-supported catalyst compared to unsupported catalyst may be attributed to homogeneous and higher dispersion on zirconia, ionic oxygen carrying capacity of zirconia. Catalyst 10%?CuO/ZrO2 showed a best conversion efficiency of 90% at a temperature as low as 265°C for CO oxidation. T50 and T100 for propylene oxidation on zirconia-supported copper oxide were 392 and 450°C, respectively.     

Preparation and characterization of Cu-Ce-La mixed oxide as water-gas shift catalyst for fuel cells application

Cu-Ce-La mixed oxides were prepared by three precipitation methods （coprecipitation, homogeneous precipitation, and deposition precipitation） with variable precipitators and characterized using X-ray diffraction, BET, temperature-programmed reduction, and catalytic reaction for the water-gas shift. The Cu-Ce-La mixed oxide prepared by coprecipitation method with NaOH as precipitator presented the highest activity and thermal stability. Copper ion substituted quadrevalent ceria entered CeO2 （111） framework was in favor of activity and thermal stability of catalyst. The crystallinity of fresh catalysts increased with the reduction process. La^3＋ or Ce^4＋ substituted copper ion entered the CeO2 framework during reduction process. The coexistence of surface copper oxide （crystalline） and pure bulk crystalline copper oxide both contributed to the high activity and thermal stability of Cu-Ce-La mixes oxide catalyst.     

亚胺基团对Schiff碱基过渡金属聚合物电化学性能的影响

采用化学合成法分别制备具有不同亚胺基团的单体Ni（salen）、Ni（salphen）,并分别配制成浓度为1 mmol/L单体溶液.在电化学工作站上,以制备好的碳纳米管电极片为工作电极,用循环伏安法进行电化学聚合得到对应的Schiff碱基过渡金属聚合物.用场发射扫描电镜观察聚合物的微观形貌,以及进行循环伏安法、恒流充放电法、交流阻抗谱测试.结果显示,聚合物poly[Ni（salen）]在碳纳米管层表面形成了带状聚合物,而聚合物poly[Ni（salphen）]沿着碳纳米管沉积生长.经测试得知：聚合物poly[Ni（salen）]和poly[Ni（salphen）]的最大电荷扩散系数分别为1.86×10-9 cm2/s、1.23×10-8 cm2/s,并且在0.05 mA/cm2下的比容量分别为66.6 F/g、106.3 F/g.具有亚胺苯环基团的Schiff 碱基过渡金属聚合物电化学性能较具有亚胺乙基基团的电化学性能更好.     

Stereochemistry of cytochrome P-450-catalyzed epoxidation and prosthetic heme alkylation

Oxidation of 1-octene by cytochrome P-450 results concurrently in formation of 1,2-oxidooctane and in N-alkylation by the catalytically activated olefin of the prosthetic heme group. The stereochemistry of trans-1-[1-2H]octene is retained during both transformations. This alkylation stereochemistry requires addition of the pyrrole nitrogen and the activated oxygen to the same side of the double bond, a reaction geometry opposite to that expected if the heme were alkylated by the epoxide metabolite. Stereochemical analysis shows that the S enantiomer of the epoxide is formed in slight excess over the R enantiomer by oxidation of the re and si faces, respectively, of the olefin, but that heme alkylation only occurs during oxidation of the re face. The stereochemical specificity of epoxidation and heme alkylation requires that (a) the two processes proceed by independent (probably concerted) mechanisms, or (b) the two processes diverge from a common acyclic intermediate.     

Novel Chiral PNNP-Ru Complexes:Synthesis and Application in Asymmetric Transfer Hydrogenation of Ketones

The efficient catalytic systems generated in situ from RuCl2(PPh3)3 and chiral ligands N,N-bis[2-(di-otolylphosphino)-benzyl]cyclohexane-1,2-diamine(2) were employed for asymmetric transfer hydrogenation of aromatic ketones, giving the corresponding optically active alcohols with high activities(up to 99% conversion) and excellent enantioselectivities(up to 96% e.e.) under mild conditions. The chiral ruthenium(Ⅱ) complex (R.,R)-3 has been prepared and characterized by NMR and X-ray crystallography.     

Nurse practitioners, practice nurses and nurse specialists: what''s in a name?

Nitric oxide (NO) readily makes corresponding complexes not only with ferrous iron but also with ferric iron. However, NO-ferric complexes of many heme proteins were unstable, while horseradish peroxidase formed the very stable NO-ferric porphyrin complex with a shift of the Soret band of the absorption wavelength from 396.5 nm to 420.0 nm. The concentration of NO in aqueous media could be monitored by measuring the absorption changes, and the detection limit was 10 nM. The simple procedure is convenient for concentration determination of NO solution.     

纳米氧化铜的制备及光催化降解亚甲基蓝

近年来,印染废水造成环境污染的问题日益严重。为处理印染废水,以亚甲基蓝(MB)为目标降解物,采用化学沉淀法制备出了纤维状和球状两种形貌的纳米氧化铜,利用XRD和SEM对样品进行表征。当催化剂投加量0.1 g、初始pH=9、亚甲基蓝初始浓度5 mg/L、质量分数为30%的H₂O₂滴加量1 mL、紫外光下反应75 min时,亚甲基蓝溶液降解率最高,且催化剂循环使用3次后,亚甲基蓝的降解率仍在88%以上,具有优良的稳定性。纳米氧化铜的形貌相同时,晶粒尺寸越小,对亚甲基蓝催化降解效果越好。相同条件下,球状纳米氧化铜催化降解亚甲基蓝的能力强于纤维状纳米氧化铜。     

Effect of CuO species and oxygen vacancies over CuO/CeO2 catalysts on low-temperature oxidation of ethyl acetate

The catalytic oxidation of ethyl acetate(EA) was studied over CuO/CeO₂ catalysts which were prepared by ball milling with different precursors(copper oxide,cerium acetate,cerium dioxide,copper acetate and cerium hydroxide).The CuO/CeO₂ catalyst(O-A) prepared with copper oxide and cerium acetate as precursors shows very high catalytic activity that 100% EA conversion is achieved at low temperature of 220℃.It is found that specific surface area(112.8 m²/g),particle...     

Differential solvation of "core" trimannoside complexes of the Dioclea grandiflora lectin and concanavalin A detected by primary solvent isotope effects in isothermal titration microcalorimetry

The thermodynamics of binding of the Man/Glc-specific seed lectin from Dioclea grandiflora (DGL) to deoxy analogs of the "core" trimannoside, 3, 6-di-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside was determined by isothermal titration microcalorimetry (ITC) in the first paper of this series (Dam, T. K., Oscarson, S., and Brewer, C. F. (1998) J. Biol. Chem. 273, 32812-32817). The data showed binding of specific hydroxyl groups on all three residues of the trimannoside, similar to that observed for ConA (Gupta, D., Dam, T. K., Oscarson, S., and Brewer, C. F. (1997) J. Biol. Chem. 272, 6388-6392). However, differences exist in the thermodynamics of binding of monodeoxy analogs of the alpha(1-6) Man residue of the trimannoside to the two lectins. The x-ray crystal structure of DGL complexed to the core trimannoside, presented in the second paper in this series (Rozwarski, D. A., Swami, B. M., Brewer, C. F., and Sacchettini, J. C. (1998) J. Biol. Chem. 273, 32818-32825), showed the overall structure of the complex to be similar to that of the ConA-trimannoside complex. Furthermore, the trimannoside is involved in nearly identical hydrogen bonding interactions in both complexes. However, differences were noted in the arrangement of ordered water molecules in the binding sites of the two lectins. The present study presents ITC measurements of DGL and ConA binding to the monodeoxy analogs of the trimannoside in hydrogen oxide (H2O) and deuterium oxide (D2O). The solvent isotope effects present in the thermodynamic binding data provide evidence for altered solvation of the parent trimannoside complexes at sites consistent with the x-ray crystal structures of both lectins. The results indicate that the differences in the thermodynamics of DGL and ConA binding to alpha(1-6) monodeoxy analogs of the trimannoside do not correlate with solvation differences of the parent trimannoside complexes.     

Spectroscopic, kinetic, and electrochemical characterization of heterologously expressed wild-type and mutant forms of copper-containing nitrite reductase from Rhodobacter sphaeroides 2.4.3

We report the development of a high-yield heterologous expression system for the copper-containing nitrite reductase from a denitrifying variant of Rhodobacter sphaeroides. Typical yields of wild-type protein are 20 mg L-1, which can be fully loaded with copper. Nitrite reductase contains an unusual blue-green Type 1 copper center with a redox/electron transfer function and a nearby Type 2 center where nitrite binds and is reduced to nitric oxide. The wild-type enzyme was characterized by: (1) its blue-green Type 1 optical spectrum; (2) its EPR spectrum showing rhombic character to its Type 1 center and nitrite perturbation to its Type 2 center; (3) its 247-mV Type 1 midpoint potential which is low relative to other Type 1 centers; and (4) its kinetics as measured by both steady-state and stopped-flow methods. The Type 2 copper reduction potential as monitored by EPR in the absence of nitrite was below 200 mV so that reduction of the Type 2 center by the Type 1 center in the absence of nitrite is not energetically favored. The mutation M182T in which the methionine ligand of Type 1 copper was changed to a threonine resulted in a blue rather than blue-green Type 1 center, a midpoint potential that increased by more than 100 mV above that of the wild-type Type 1 center, and a somewhat reduced nitrite reductase activity. The blue color and midpoint potential of M182T are reminiscent of plastocyanin, but the Type 1 cupric HOMO ground-state electronic g value and copper hyperfine properties of M182T (as well as cysteine and histidine ENDOR hyperfine properties; see next paper) were unchanged from those of the blue-green native Type 1 center. His287 is a residue in the Type 2 region whose imidazole ring was thought to hydrogen bond to the Type 2 axial ligand but not directly to Type 2 copper. The mutation H287E resulted in a 100-fold loss of enzyme activity and a Type 2 EPR spectrum (as well as ENDOR spectra; see next paper) which were no longer sensitive to the presence of nitrite.     

Characterization of atherosclerotic plaque components by high resolution quantitative MR and US imaging

Dopamine beta-monooxygenase requires copper ions for catalytic activity. The stoichiometry of copper activation has been a matter of discussion, but most of the recent literature agrees on a model with two copper ions per active site. We have now reinvestigated this problem with kinetic experiments at high and low protein levels. The apoenzyme (metal free) is rapidly activated by adding copper. Incremental addition of copper to high levels (up to 10 microM subunits) of enzyme raised the catalytic activity until the stoichiometric relationship between copper and enzyme subunits was 1:1. No increase in activity was observed upon addition of copper in excess of this up to levels of 3 Cu/subunit. Experiments at low protein levels (0.12 microM subunits) revealed that copper activation is described by a hyperbolic, Michaelis-Menten-type curve. This is to be expected for the 1 Cu/subunit model, whereas the 2/1 model predicts sigmoid curves. With an incremental addition of apoenzyme (high level) to a fixed level of copper, a sharp break was again observed at 1 Cu/subunit, and excess apoenzyme showed no evidence of the inhibition predicted by the 2 Cu/subunit model. Steady-state kinetics experiments with variation of the concentrations of copper and the three substrates supported an equilibrium-ordered mechanism, whereby a single activating copper ion is trapped in the active site by the substrates. Treatment of enzyme containing more than 1 Cu/subunit [both the Cu(I) and Cu(II) states were examined] with a chelating column resulted in loss of all copper in excess of 1 Cu/subunit. Reactivation of apoenzyme by vanadyl ions was studied, both as dopamine beta-monooxygenase-catalyzed electron transfer and hydroxylation. The maximal velocity with vanadium was 70% of that with copper, and the activation curve was clearly hyperbolic, again supporting the requirement of only one metal ion per active site. In conclusion, our results support the view that the copper ions bound to dopamine beta-monooxygenase in excess of 1 Cu/subunit are not required for catalysis.     

Cerium (Ⅳ) oxide nanocomposites:Catalytic properties and industrial application

This review article provides a new insight on the application of cerium oxide(CeO_2)-metal oxide nanocomposites as catalyst with enhanced reducibility and improved oxygen(O_2) storage capacity,especially in the varying chemical reaction processes including combustion,oxidation,epoxidation and redox.The CeO_2-metal oxide interaction plays an important role in controlling particle size,O_2 availability and coke resistance properties of the catalyst.Strong metal oxide-CeO_2 interaction also assists in generating small and highly dispersed particles,resists sintering of catalyst particles during the catalysis process,and consequently improves the O_2 availability of the catalyst.Indeed,CeO_2 not only provides an active support to heterogeneous catalyst,but also creates a new active site at the metal-support interface to produce stronger nanoparticle bonding through the surface O_2 vacancy.This consequently produces a heterogeneous catalytic system with promising reaction rate and catalytic stability in many industrial applications such as CO_2 hydrogenation,CO oxidatio n,biodiesel productio n,gas reduction,photocatalytic and methane steam reforming.     

MnPcS4: a new MRI contrast enhancing agent for tumor localisation in mice

Manganese-tetrasulfonated phthalocyanine (MnPcS4) has been evaluated as a potential contrast agent in Magnetic resonance imaging (MRI) for tumor localisation in mice. MnPcS4 showed favourable molar relaxivity, much better than Gd-DTPA and comparable to tetrasulfonated manganese complex of porphyrin (TPPS4). Tumors showed selective retention of the metal complex (dye) with the peak value reached at 24 hours following intravenous administration. Dye concentration in tumors remained consistently higher than either kidney or muscle tissue both at 1 and 24 hours and a 10-fold increase in tumor-to-muscle ratio over the control was seen at 24 hr. Normal liver tissue, however, showed higher concentration than tumor at all times during the study. A linear correlation was found between longitudinal relaxation rate (1/T1) and the corresponding concentration of MnPcS4 in various tissues. MR imaging done in animals using 1.5 T superconducting clinical imager showed a mean percent increase in signal intensity of 131.8% (SD +/- 32.86) in the tumor and a 70% increase in tumor-to-muscle ratio over the pretreatment value, at 24 hr. The results suggest that MnPcS4 is a potential tumor-selective contrast agent in MRI.     

Effect of tungsten oxide on ceria nanorods to support copper species as CO oxidation catalysts

In this work,tungsten oxide with different concentrations(0,0.4 at%,2.0 at% and 3.2 at%) was introduced to the ceria nanorods via a deposition-precipitation(DP) approach,and copper species of ca.10 at% were sequentially anchored onto the modified ceria support by a similar DP route.The aim of the study was to investigate the effect of the amount of tungsten oxide(0,0.4 at%,2.0 at%,and 3.2 at%) modifier on the copper-ceria catalysts for CO oxidation reaction and shed light on the structure-activity relationship.By the aids of multiple characterization techniques including N_2 adsorption,high-resolution transmission electron microscopy(HRTEM),powder X-ray diffraction(XRD),X-ray absorption fine structure(XAFS),and temperature-programmed reduction by hydrogen(H_2-TPR) in combination with the catalytic performance for CO oxidation reaction,it is found that the copper-ceria samples maintain the crystal structure of the fluorite fcc CeO_2 phase with the same nanorod-like morphology with the introduction of tungsten oxide,while the textural properties(the surface area,pore volume and pore size) of ceria support and copper-ceria catalysts are changed,and the oxidation states of copper and tungsten are kept the same as Cu~(2+)and W~(6+)before and after the reaction,but the introduction of tungsten oxide(WO_3)significantly changes the metal-support interaction(transfer the CuO_x clusters to Cu-[O_x]-Ce species),which delivers to impair the catalytic activity of copper-ceria catalysts for CO oxidation reaction.     

Structure of the Fusarium oxysporum endoglucanase I with a nonhydrolyzable substrate analogue: substrate distortion gives rise to the preferred axial orientation for the leaving group

Endoglucanase I (EG I) is a cellulase, from glycosyl hydrolase family 7, which cleaves the beta-1,4 linkages of cellulose with overall retention of configuration. The structure of the EG I from Fusarium oxysproum, complexed to a nonhydrolyzable thiooligosaccharide substrate analogue, has been determined by X-ray crystallography at a resolution of 2.7 A utilizing the 4-fold noncrystallographic symmetry present in the asymmetric unit. The electron density map clearly reveals the presence of three glucosyl units of the inhibitor, consistent with the known number of sugar-binding subsites, located at the active site of the enzyme in the -2, -1, and +1 subsites, i.e., actually spanning the point of enzymatic cleavage. The pyranose ring at the point of potential enzymatic cleavage is clearly distorted from the standard 4C1 chair as was originally suggested for beta-retaining enzymes by Phillips [Ford, L.O., Johnson, L.N., Machin, P. A., Phillips, D.C., & Tijan, T. (1974) J. Mol. Biol, 88, 349-371]. The distortion observed goes beyond the "sofa" conformation observed in previous studies and results in a conformation whose salient feature is the resulting quasi-axial orientation for the glycosidic bond and leaving group, as predicted by stereoelectronic theory. An almost identical conformation has recently been observed in a complex of chitobiase with its unhydrolyzed substrate [Tews, I., Perrakis, A., Oppenheim, A., Dauter, Z., Wilson, K. S., & Vorgias, C. E. (1996) Nat. Struct. Biol. 3, 638-648]. The striking similarity between these two complexes extends beyond the almost identical pyranose ring distortion. The overlap of the two respective sugars places the enzymatic nucleophile of endoglucanase I in coincidence with the C2 acetamido oxygen of N-acetylglucosamine in the catalytic site of the chitobiase, substantiating the involvement of this group in the catalytic mechanism of chitobiase and related chitinolytic enzymes. The endoglucanase I complex with the thiosaccharide substrate analogue clearly illustrates the potential of nonhydrolyzable sulfur-linked oligosaccharides in the elucidation of substrate binding and catalysis by glycosyl hydrolases.