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Parasporin-2 is a protein toxin that is isolated from parasporal inclusions of the Gram-positive bacterium Bacillus thuringiensis. Although B. thuringiensis is generally known as a valuable source of insecticidal toxins, parasporin-2 is not insecticidal, but has a strong cytocidal activity in liver and colon cancer cells. The 37-kDa inactive nascent protein is proteolytically cleaved to the 30-kDa active form that loses both the N-terminal and the C-terminal segments. Accumulated cytological and biochemical observations on parasporin-2 imply that the protein is a pore-forming toxin. To confirm the hypothesis, we have determined the crystal structure of its active form at a resolution of 2.38 A. The protein is unusually elongated and mainly comprises long beta-strands aligned with its long axis. It is similar to aerolysin-type beta-pore-forming toxins, which strongly reinforce the pore-forming hypothesis. The molecule can be divided into three domains. Domain 1, comprising a small beta-sheet sandwiched by short alpha-helices, is probably the target-binding module. Two other domains are both beta-sandwiches and thought to be involved in oligomerization and pore formation. Domain 2 has a putative channel-forming beta-hairpin characteristic of aerolysin-type toxins. The surface of the protein has an extensive track of exposed side chains of serine and threonine residues. The track might orient the molecule on the cell membrane when domain 1 binds to the target until oligomerization and pore formation are initiated. The beta-hairpin has such a tight structure that it seems unlikely to reform as postulated in a recent model of pore formation developed for aerolysin-type toxins. A safety lock model is proposed as an inactivation mechanism by the N-terminal inhibitory segment.

Understanding the structural basis of thermostability and thermoactivity, and their interdependence, is central to the successful future exploitation of extremophilic enzymes in biotechnology. However, the structural basis of thermostability is still not fully characterized. Ionizable residues play essential roles in proteins, modulating protein stability, folding and function. The dominant roles of the buried polar contacts and ion pairs have been reviewed by distinguishing between the inside polar contacts and the total intramolecular polar contacts, and by evaluating their contribution as molecular determinants for protein stability using various protein structures from hyperthermophiles, thermophiles and mesophilic organisms. The analysis revealed that the remarkably increased number of internal polar contacts in a monomeric structure probably play a central role in enhancing the melting temperature value up to 120 degrees C for hyperthermophilic enzymes from the genus Pyrococcus. These results provide a promising contribution for improving the thermostability of enzymes by modulating buried polar contacts and ion pairs.

The X-ray crystal structure of RadB from Thermococcus kodakaraensis KOD1, an archaeal homologue of the RecA/Rad51 family proteins, have been determined in two crystal forms. The structure represents the core ATPase domain of the RecA/ Rad51 proteins. Two independent molecules in the type 1 crystal were roughly related by 7-fold screw symmetry whereas non-crystallographic 2-fold symmetry was observed in the type 2 crystal. The dimer structure in the type 1 crystal is extended to construct a helical assembly, which resembles the filamentous structures reported for other RecA/Rad51 proteins. The molecular interface in the type 1 dimer is formed by facing a basic surface patch of one monomer to an acidic one of the other. The empty ATP binding pocket is located at the interface and barely concealed from the outside similarly to that in the active form of the RecA filament. The model assembly has a positively charged belt on one surface bordering the helical groove suitable for facile binding of DNA. Electron microscopy has revealed that, in the absence of ATP and DNA, RadB forms a filament with a similar diameter to that of the hypothetical assembly, although its helical properties were not confirmed.

The reaction center-light-harvesting 1 (RC-LH1) core complex is the photosynthetic apparatus in the membrane of the purple photosynthetic bacterium Rhodopseudomonas viridis. The RC is surrounded by an LH1 complex that is constituted of oligomers of three types of apoproteins (alpha, beta and gamma chains) with associated bacteriochlorophyll bs and carotenoid. It has been crystallized by the sitting-drop vapour-diffusion method. A promising crystal diffracted to beyond 8.0 A resolution. It belonged to space group P1, with unit-cell parameters a = 141.4, b = 136.9, c = 185.3 A, alpha = 104.6, beta = 94.0, gamma = 110.7 degrees. A Patterson function calculated using data between 15.0 and 8.0 A resolution suggested that the LH1 complex is distributed with quasi-16-fold rotational symmetry around the RC.

Maltohexaose-producing amylase, called G6-amylase (EC 3.2.1.98), from alkalophilic Bacillus sp.707 predominantly produces maltohexaose (G6) from starch and related alpha-1,4-glucans. To elucidate the reaction mechanism of G6-amylase, the enzyme activities were evaluated and crystal structures were determined for the native enzyme and its complex with pseudo-maltononaose at 2.1 and 1.9 A resolutions, respectively. The optimal condition for starch-degrading reaction activity was found at 45 degrees C and pH 8.8, and the enzyme produced G6 in a yield of more than 30% of the total products from short-chain amylose (DP = 17). The crystal structures revealed that Asp236 is a nucleophilic catalyst and Glu266 is a proton donor/acceptor. Pseudo-maltononaose occupies subsites -6 to +3 and induces the conformational change of Glu266 and Asp333 to form a salt linkage with the N-glycosidic amino group and a hydrogen bond with secondary hydroxyl groups of the cyclitol residue bound to subsite -1, respectively. The indole moiety of Trp140 is stacked on the cyclitol and 4-amino-6-deoxyglucose residues located at subsites -6 and -5 within a 4 A distance. Such a face-to-face short contact may regulate the disposition of the glucosyl residue at subsite -6 and would govern the product specificity for G6 production.

A triclinic crystal of hen egg-white lysozyme obtained from a D2O solution at 313 K was transformed into a new triclinic crystal by slow release of solvent under a temperature-regulated nitrogen-gas stream. The progress of the transition was monitored by X-ray diffraction. The transition started with the appearance of strong diffuse streaks. The diffraction spots gradually fused and faded with the emergence of diffraction from the new lattice; the scattering power of the crystal fell to a resolution of 1.5 A from the initial 0.9 A resolution. At the end of the transition, the diffuse streaks disappeared and the scattering power recovered to 1.1 A resolution. The transformed crystal contained two independent molecules and the solvent content had decreased to 18% from the 32% solvent content of the native crystal. The structure was determined at 1.1 A resolution and compared with the native structure refined at the same resolution. The backbone structures of the two molecules in the transformed crystal were superimposed on the native structure with root-mean-square deviations of 0.71 and 0.96 A. A prominent structural difference was observed in the loop region of residues Ser60-Leu75. In the native crystal, a water molecule located at the centre of this helical loop forms hydrogen bonds to main-chain peptide groups. In the transformed crystal, this water molecule is replaced by a sodium ion with octahedral coordination that involves water molecules and a nitrate ion. The peptide group connecting Arg73 and Asn74 is rotated by 180 degrees so that the CO group of Arg73 can coordinate to the sodium ion. The change in the X-ray diffraction pattern during the phase transition suggests that the transition proceeds at the microcrystal level. A mechanism is proposed for the crystal transformation.

DNA primases are essential components of the DNA replication apparatus in every organism. Reported here are the biochemical characteristics of a thermostable DNA primase from the thermophilic archaeon Pyrococcus horikoshii, which formed the oligomeric unit L(1)S(1) and synthesized long DNA primers 10 times more effectively than RNA primers. The N-terminal (25KL) and C-terminal halves (20KL) of the large subunit (L) play distinct roles in regulating de novo DNA synthesis of the small catalytic subunit (S). The 25KL domain has a dual function. One function is to depress the large affinity of the intrasubunit domain 20KL for the template DNA until complex (L(1)S(1) unit) formation. The other function is to tether the L subunit tightly to the S subunit, probably to promote effective interaction between the intrasubunit domain 20KL and the active center of the S subunit. The 20KL domain is a central factor to enhance the de novo DNA synthesis activity of the catalytic S subunit since the total affinity of the L(1)S(1) unit is mainly derived from the affinity of 20KL, which is elevated more than 10 times by the heterodimer formation, presumably due to the cancellation of the inhibitory activity of 25KL through tight binding to the S subunit.

In spite of the belonging to the same c-type lysozyme family, hen egg-white lysozyme (HEWL) was much less susceptible to the dual-affinity labeling with 2′,3′-epoxypropyl β-glycoside of N-acetyllactosamine (Galβ1,4GlcNAc-Epo) than human lysozyme (HL). The three-dimensional structures of the HEWL labeled with single Galβ1,4GlcNAc-Epo and the Glu102-mutant HL labeled with double Galβ1,4GlcNAc-Epo were determined by X-ray crystallography at resolutions of 1.85 and 2.0 A, respectively. The overall conformation and the interaction mode of the carbohydrate ligand part in the singly labeled HEWL and the doubly labeled Glu102-mutant HL were basically identical to those of the correspondingly labeled wild-type HL with minor alterations in some stereochemical parameters. A detailed comparison of the structures revealed the key protein–carbohydrate and carbohydrate–carbohydrate interactions essential for the dual labeling. It was suggested that the difference in the efficiency of the dual labeling was caused by the structural difference between Gln104 in HL and Asn103 in HEWL. The relevance to our previous study and the carbohydrate–carbohydrate interaction on cell-surface membranes were discussed. Copyright © 2003 John Wiley & Sons, Ltd.

The crystal structure of turkey egg lysozyme (TEL) complexed with di-N-acetylchitobiose (NAG2) was refined at 1.19 A resolution by the full-matrix least-squares method with anisotropic temperature factors, and its thermal motion was evaluated by the TLS method. The average ESDs of atomic parameters of nonhydrogen atoms were 0.030 A for coordinates and 0.025 A2 for anisotropic temperature factors. The active site cleft of TEL binds the α-anomer of NAG2 in a nonproductive binding mode with its pyranose rings parallel to a β-sheet. The TEL structure was compared with the re-refined 1.12 A structure of native TEL. The RMS difference for equivalent Cα atoms was 0.103 A and a relatively large difference was observed in the region of residues 104–125 rather than in the β-sheet region where NAG2 was bound. In contrast, the temperature factor of the β-sheet region was significantly decreased by the NAG2 binding. The TLS model that describes the rigid body motion in translation, libration, and screw motion was adopted for the evaluation of the molecular motion of TEL and NAG2, and the TLS parameters were determined by the least-squares fit to Uij. The contribution of the external motion of TEL was estimated to be 55.8% of the observed temperature factor for the native structure and 45.9% for the NAG2 complex. The internal motion of TEL represented with atomic thermal ellipsoids was very similar between the native and complex structures except the NAG2 binding region. In the structure of NAG2, the rigid body motion dominates the thermal motion. The center of rotation of NAG2, 4.45A far from the center of gravity, is on the nitrogen atom of the acetylamino group that is hydrogen bonded to the main-chain peptide groups of Asn49 and Ala107. The rigid body motion of NAG2 indicates that the acetylamino group is most strongly bound to the active site, and the recognition of this group is a crucial step of the substrate binding. Proteins 2002;48:53–62. © 2002 Wiley-Liss, Inc.

The interactions of wheat-germ agglutinin (WGA) with the GlcNAc beta 1,6Gal sequence, a characteristic component of branched poly-N-acetyllactosaminoglycans, were investigated using isothermal titration calorimetry and X-ray crystallography. GlcNAc beta 1,6Gal exhibited an affinity greater than GlcNAc beta 1,4GlcNAc to all WGA isolectins, whereas Gal beta 1,6GlcNAc showed much less affinity than GlcNAc beta 1,4GlcNAc. X-ray structural analyses of the glutaraldehyde-crosslinked WGA isolectin 3 crystals in complex with GlcNAc beta 1,6Gal, GlcNAc beta 1,4GlcNAc and GlcNAc beta 1,6Gal beta 1,4Glc were performed at 2.4, 2.2 and 2.2 A resolution, respectively. In spite of different glycosidic linkages, GlcNAc beta 1,6Gal and GlcNAc beta 1,4GlcNAc exhibited basically similar binding modes to each other, in contact with side chains of two aromatic residues, Tyr64 and His66. However, the conformations of the ligands in the two primary binding sites were not always identical. GlcNAc beta 1,6Gal showed more extensive variation in the parameters defining the glycosidic linkage structure compared to GlcNAc beta 1,4GlcNAc, demonstrating large conformational flexibility of the former ligand in the interaction with WGA. The difference in the ligand binding conformation was accompanied by alterations of the side chain conformation of the amino acid residues involved in the interactions. The hydrogen bond between Ser62 and the non-reducing end GlcNAc was always observed regardless of the ligand type, indicating the key role of this interaction. In addition to the hydrogen bonding and van der Waals interactions, CH--pi interactions involving Tyr64, His66 and Tyr73 are suggested to play an essential role in determining the ligand binding conformation in all complexes. One of the GlcNAc beta 1,6Gal ligands had no crystal packing contact with another WGA molecule, therefore the conformation might be more relevant to the interaction mode in solution.

Ultica dioica agglutinin, a plant lectin from the stinging nettle, consists of a total of seven individual isolectins. One of these structures, isolectin I, was determined at 1.9 A resolution by the X-ray method. The crystals belong to the space group P2(1) and the asymmetric unit contains two molecules related by local twofold symmetry. The molecule consists of two hevein-like chitin-binding domains lacking distinct secondary structure, but four disulfide bonds in each domain maintain the tertiary structure. The backbone structure of the two independent molecules is essentially identical and this is similarly true of the sugar-binding sites. In the crystal, the C-terminal domains bind Zn(2+) ions at the sugar-binding site. Owing to their location near a pseudo-twofold axis, the two zinc ions link the two independent molecules in a tail-to-tail arrangement: thus, His47 of molecule 1 and His67 of molecule 2 coordinate the first zinc ion, while the second zinc ion links Asp75 of molecule 1 and His47 of molecule 2.

Crystals have been grown of the V(1)-ATPase sector of the V-type ATP synthase complex (V(0)V(1)) from the thermophilic eubacterium Thermus thermophilus HB8. These crystals are grown by the vapor diffusion method in the presence of 5 mM Mg-ADP, from solutions containing 100 mM sodium acetate and 2 M sodium formate, pH 5.5. The crystals diffracted X rays beyond 3.4 A in resolution on a synchrotron radiation source. The crystals belong to the trigonal space group P3, with unit cell dimensions of a = b = 89.0 A, c = 179.2 A, and gamma = 120 degrees. The unit cell presumably contains one molecule of V(1)-ATPase and the V(m) value is calculated as 3.0 A(3)/Da.

Crystal structures of four 6-monosubstituted β-cyclodextrins, 6-deoxy-6-(1-propyl)amino-β-cyclodextrin (1), 6-deoxy-6-[(R)-1-cyclohexylethyl]amino-β-cyclodextrin (2), 6-deoxy-6-[(R)-1-phenylethyl]amino-β-cyclodextrin (3), and 6-deoxy-6-[(1R,2S)-2-hydroxyindan-1-yl]amino-β-cyclodextrin (4) were determined by X-ray analysis. In each crystal, the substituent group is inserted into the adjacent β-cyclodextrin ring from the secondary hydroxy side. This donor–acceptor type self-association through intermolecular inclusion generates a one-dimensional polymeric chain. Two types of arrangement of the β-cyclodextrin rings, a helical form and a linear form, were observed. Compounds 1 and 2 form a helically extended chain along a crystallographic twofold screw axis. In contrast the β-cyclodextrin rings are linearly stacked in the crystals of 3 and 4. The crystal packing of 3 is similar to that of a channel-type structure, while the arrangement of the β-cyclodextrin ring of 4 belongs to the cage-type. These crystal structures suggest that the self-assembly of the 6-monosubstituted β-cyclodextrins is regulated by the physical and chemical properties of the substituent group included in the adjacent β-cyclodextrin ring. A β-cyclodextrin ring with a linear and/or flexible substituent group tends to produce twofold helical packing. The inclusion of a planar and rigid group imposes restrictions on the relative orientation of the macrocycle and this causes variation in the one-dimensional arrangement depending on the shape, size, and orientation of the substituent group.

We determined the crystal structure of the liganded form of alpha-aminotransferase from a hyperthermophile, Pyrococcus horikoshii. This hyperthermophilic enzyme did not show domain movement upon binding of an acidic substrate, glutamate, except for a small movement of the alpha-helix from Glu16 to Ala25. The omega-carboxyl group of the acidic substrate was recognized by Tyr70* without its side-chain movement, but not by positively charged Arg or Lys. Compared with the homologous enzymes from Thermus thermophilus HB8 and Escherichia coli, it was suggested that the more thermophilic the enzyme is, the smaller the domain movement is. This rule seems to be applicable to many other enzymes already reported.

Heptakis(2,6-di-O-ethyl)-beta-cyclodextrin (DE-beta-CD) was crystallized in two forms from hexane and 95% aqueous methanol, respectively: A form I crystal with the space group P2(1)2(1)2(1) and a form II crystal with the space group P3(1). In both crystals, DE-beta-CD molecules are in a round shape with intramolecular O-3-H...O-2 hydrogen bonds. In the form I crystal, the DE-beta-CD molecules are arranged along the twofold screw axis to form a helically extended polymeric chain by including the 6-O-ethyl groups of the adjacent molecule. One hexane molecule with twofold disorder is located in the intermolecular channel along the a-axis. In contrast, the DE-beta-CD molecules in the form II crystal form a helical arrangement along the threefold screw axis. One methanol and one water molecule are included on the O-6 side of the molecular cavity. The water molecule links the methanol molecule and two ethoxy groups of the adjacent DE-beta-CD molecule with hydrogen bonds. The result suggests the important role of solvent in the formation of helical arrangement of DE-beta-CD molecules.

Chemically prepared hevein domains (HDs), N-terminal domain of an antifungal protein from Nicotiana tabacum (CBP20-N) and an antimicrobial peptide from Amaranthus caudatus (Ac-AMP2), were examined for their affinity for chitin, a beta-1,4-linked polymer of N-acetylglucosamine. An intact binding domain, CBP20-N, showed a higher affinity than a C-terminal truncated domain, Ac-AMP2. The formation of a pyroglutamate residue from N-terminal Gln of CBP20-N increased the affinity. The single replacement of any aromatic residue of Ac-AMP2 with Ala resulted in a significant reduction in affinity, suggesting the importance of the complete set of three aromatic residues in the ligand binding site. The mutations of Phe18 of Ac-AMP2 to the residues with larger aromatic rings, i.e. Trp, beta-(1-naphthyl)alanine or beta-(2-naphthyl)alanine, enhanced the affinity, whereas the mutation of Tyr20 to Trp reduced the affinity. The affinity of an HD for chitin might be improved by adjusting the size and substituent group of stacking aromatic rings.

Urtica dioica agglutinin is a small plant lectin that binds chitin. We purified the isolectin VI (UDA-VI) and crystal structures of the isolectin and its complex with tri-N-acetylchitotriose (NAG3) were determined by X-ray analysis. The UDA-VI consists of two domains analogous to hevein and the backbone folding of each domain is maintained by four disulfide bridges. The sequence similarity of the two domains is not high (42 %) but their backbone structures are well superimposed except some loop regions. The chitin binding sites are located on the molecular surface at both ends of the dumbbell-shape molecule. The crystal of the NAG3 complex contains two independent molecules forming a protein-sugar 2:2 complex. One NAG3 molecule is sandwiched between two independent UDA-VI molecules and the other sugar molecule is also sandwiched by one UDA-VI molecule and symmetry-related another one. The sugar binding site of N-terminal domain consists of three subsites accommodating NAG3 while two NAG residues are bound to the C-terminal domain. In each sugar-binding site, three aromatic amino acid residues and one serine residue participate to the NAG3 binding. The sugar rings bound to two subsites are stacked to the side-chain groups of tryptophan or histidine and a tyrosine residue is in face-to-face contact with an acetylamino group, to which the hydroxyl group of a serine residue is hydrogen-bonded. The third subsite of the N-terminal domain binds a NAG moiety with hydrogen bonds. The results suggest that the triad of aromatic amino acid residues is intrinsic in sugar binding of hevein-like domains.

The product specificity of cyclodextrin glucanotransferase (CGTase) from alkalophilic Bacillus sp. #1011 is improved to near-uniformity by mutation of histidine-233 to asparagine. Asparagine 233-replaced CGTase (H233N-CGTase) no longer produces alpha-cyclodextrin, while the wild-type CGTase from the same bacterium produces a mixture of predominantly alpha-, beta-, and gamma-cyclodextrins, catalyzing the conversion of starch into cyclic or linear alpha-1,4-linked glucopyranosyl chains. In order to better understand the protein engineering of H233N-CGTase, the crystal structure of the mutant enzyme complexed with a maltotetraose analog, acarbose, was determined at 2.0 A resolution with a final crystallographic R value of 0.163 for all data. Taking a close look at the active site cleft in which the acarbose molecule is bound, the most probable reason for the improved specificity of H233N-CGTase is the removal of interactions needed to form a compact ring like a-cyclodextrin.

Aromatic amino acid aminotransferase (ArATPh), which has a melting temperature of 120 degrees C, is one of the most thermostable aminotransferases yet to be discovered. The crystal structure of this aminotransferase from the hyperthermophilic archaeon Pyrococcus horikoshii was determined to a resolution of 2.1 A. ArATPh has a homodimer structure in which each subunit is composed of two domains, in a manner similar to other well characterized aminotransferases. By the least square fit after superposing on a mesophilic ArAT, the ArATPh molecule exhibits a large deviation of the main chain coordinates, three shortened alpha-helices, an elongated loop connecting two domains, and a long loop transformed from an alpha-helix, which are all factors that are likely to contribute to its hyperthermostability. The pyridine ring of the cofactor pyridoxal 5'-phosphate covalently binding to Lys(233) is stacked parallel to F121 on one side and interacts with the geminal dimethyl-CH/pi groups of Val(201) on the other side. This tight stacking against the pyridine ring probably contributes to the hyperthermostability of ArATPh. Compared with other ArATs, ArATPh has a novel substrate specificity, the order of preference being Tyr > Phe > Glu > Trp > His>> Met > Leu > Asp > Asn. Its relatively weak activity against Asp is due to lack of an arginine residue corresponding to Arg(292)* (where the asterisk indicates that this is a residues supplied by the other subunit of the dimer) in pig cytosolic aspartate aminotransferase. The enzyme recognizes the aromatic substrate by hydrophobic interaction with aromatic rings (Phe(121) and Tyr(59)*) and probably recognizes acidic substrates by a hydrophilic interaction involving a hydrogen bond network with Thr(264)*.

The effect of the alkyl chain on the thermal stability of crystals for a series of glycolipids, methyl 6-O-alkanoylglycopyranosides, was investigated using thermal analysis, X-ray structures and molecular mechanics. Glycolipids with two types of sugar moieties, α-galactopyranoside and β-glucopyranoside carrying an alkyl chain with various numbers of carbon atoms, 1–11, and 1–13, respectively, were used for the analyses. All the crystals have bilayer structures with interdigitated alkyl chains. The crystal packing is significantly affected by the sugar moieties but the same for individual sugar derivatives for a variety of alkyl chain lengths. Alkyl chains with an even number of carbon atoms in the α-galactoside derivatives have temperature factors (thermal parameters) which are higher than those for alkyl chains with an odd number of carbon atoms. Alkyl chains with an even number of carbon atoms in the β-glucosides have a disordered structure with two alternate conformations while the remaining derivatives have well ordered alkyl chains. The mean lengths of the hydrogen bonds in the β-glucosides with an even number of carbon atoms in the alkyl chain are longer than those with an odd number of carbon atoms, but a slight difference only is observed in the α-galactosides. The thermal stability of the α-galactosides with an odd number of carbon atoms in the alkyl chain is higher than that of α-galactosides with an even number of carbon atoms. On the other hand, the opposite behavior is observed for the β-glucosides. The difference in the melting points between β-glucosides with an odd and even number of carbon atoms in the alkyl chain is greater than the difference for the corresponding α-galactosides. The difference in the thermal stability is suggested to be related to the packing of the sugar moieties in the crystal based on molecular mechanics calculations.

The crystal structure of asparagine 233-replaced cyclodextrin glucanotransferase from alkalophilic Bacillus sp. 1011 was determined at 1.9 A resolution. While the wild-type CGTase from the same bacterium produces a mixture of mainly alpha-, beta- and gamma-cyclodextrins, catalyzing the conversion of starch into cyclic or linear alpha-1,4-linked glucopyranosyl chains, site-directed mutation of histidine-233 to asparagine changed the nature of the enzyme such that it no longer produced alpha-cyclodextrin. This is a promising step towards an industrial requirement, i.e. unification of the products from the enzyme. Two independent molecules were found in an asymmetric unit, related by pseudo two-fold symmetry. The backbone structure of the mutant enzyme was very similar to that of the wild-type CGTase except that the position of the side chain of residue 233 was such that it is not likely to participate in the catalytic function. The active site cleft was filled with several water molecules, forming a hydrogen bond network with various polar side chains of the enzyme, but not with asparagine-233. The differences in hydrogen bonds in the neighborhood of asparagine-233, maintaining the architecture of the active site cleft, seem to be responsible for the change in molecular recognition of both substrate and product of the mutant CGTase.

Crystal structure of a 1:2 complex of hexakis(3-O-acetyl-2,6-di-O-methyl)-α-cyclodextrin (ADMACD) with butylacetate was determined by the X-ray method. The space group of the crystal is P212121 with Z = 4 and D x = 1.293 g cm−3, and the cell dimensions are a = 11.087(2), b = 23.543(3), and c = 31.739(6) A. The structure was solved by the direct method and refined to the R-value of 0.123 for all the 4993 observed reflections with 1

An Fe(III)–azido complex with the formula [Fe(cyclam)(N3)2]ClO4 (cyclam=1,4,8,11-tetraazacyclotetradecane) has been synthesized from the reaction of cis-[Fe(cyclam)Cl2]Cl with sodium azide in methanol. The X-ray structural analysis reveals that the Fe(III) atom possesses a tetragonally compressed octahedral geometry with a trans configuration of two azido ions. Variable-temperature (4.5–295 K) magnetic susceptibility measurements show that the complex is low spin over the whole temperature range. 57Fe Mossbauer spectral measurements also suggest the same spin state of the Fe(III) ion.

The low temperature form of human α-lactalbumin (HAL) was crystallized from a 2 H 2 O solution and its structure was refined to the R value of 0.119 at 1.15 A resolution by the full-matrix least-squares method. Average estimated standard deviations of atomic parameters for non-hydrogen atoms were 0.038 A for coordinates and 0.044 A 2 for anisotropic temperature factors ( U ij ). The magnitude of equivalent isotropic temperature factors ( U eqv ) was highly correlated with the distance from the molecular centroid and fitted to a quadratic equation as a function of atomic coordinates. The atomic thermal motion was rather isotropic in the core region and the anisotropy increased towards the molecular surface. The statistical analysis revealed the out-of-plane motion of main-chain oxygen atoms, indicating that peptide groups are in rotational vibration around a C α ⋯C α axis. The TLS model, which describes the rigid-body motion in terms of translation, libration, and screw motions, was adopted for the evaluation of the molecular motion and the TLS parameters were determined by the least-squares fit to U ij . The reproduced U eqv cal from the TLS parameters was in fair agreement with observed U eqv , but differences were found in regions of residues, 5–22, 44–48, 70–75, and 121–123, where U eqv was larger than U eqv cal because of large local motions. To evaluate the internal motion of HAL, the contribution of the rigid-body motion was determined to be 42.4 % of U eqv in magnitude, which was the highest estimation to satisfy the condition that the U ij int tensors of the internal motion have positive eigen values. The internal motion represented with atomic thermal ellipsoids clearly showed local motions different from those observed in chicken-type lysozymes which have a backbone structure very similar to HAL. The result indicates that the internal motion is closely related to biological function of proteins.

Crystal structures of turkey and human lysozymes were refined at atomic resolution by full-matrix least-squares method with anisotropic temperature factors. The refinement converged at the R-value of 0.104 for turkey lysozyme and 0.115 for human lysozyme at 1.12 A and 1.15 A resolution, respectively, and the estimated r.m.s. coordinate errors for respective pro-teins were 0.031 A and 0.034 A. The magnitude and the degree of anisotropy of the atomic thermal motion have strong positive correlation with the square of distance from the molecular centroid. The statistical analysis suggested that such characteristics of anisotropic thermal motion are ascribed to the rigid-body rotation and local motions rather than the breathing motion.

Crystal structures of sodium, potassium and cesium salts of anionic surfactants, methyl 2-sulfoalkanoates, were analyzed by X-ray methods to characterize physico-chemical properties of the solid state in relation to counter ions. The crystal contains racemic molecules with the stereogenic β carbon atom. The crystals have a bilayer structure with the interdigitated alkyl chain of anions, while the cations and water molecules are intercalated between the layers. These crystals have different thermal stability indicated by the decrease in melting temperature in the order of potassium, cesium and sodium salts. The crystals of sodium, potassium and cesium salts contain two, one and one water molecules, respectively. The space group is Pbca for all of these crystals having the same type of crystal packing of anions regardless of the different cations. The crystal packing of the potassium salts is not significantly affected by the alkyl chain length, except for the difference in the c dimension. The energetic difference of the crystal structures was analyzed by molecular mechanics calculations using X-ray coordinates. The thermal stability of the crystal is related to the crystal structure, especially to the packing of cations and sulfonato groups between the layers. The potassium ion contributes more to the thermal stabilization of the crystal than the sodium and cesium ions because of more effective contact with the sulfonato groups by less coordination with the water molecule and by acquired electrostatic potential. The close packing of ionic layers observed in the crystals of potassium salts causes dense packing of the alkyl chains which stabilizes the crystal packing.

The crystal structure and molecular recognition behaviour of a new chiral-amino cyclodextrin are reported; van der Waals interaction, hydrogen bond and the electrostatic interactions play an important role in the self-assembling process and chiral recognition for (R)-(-)-and (S)-(+)-mandelic acid.

Crystal structure of i-cyclodextrin consisting of 14 glucose units were determined by the X-ray method. The crystal of 9 hydrate belongs to a monoclinic space group C2 with Z=2 and cell dimensions are a = 36.536(9), b = 10.085(4), c = 20.944(2) A, and β = 114.97(2)°. The molecule with crystallographic twofold symmetry is in a saddle-like shape where the elliptical macrocyclic ring is bent by 75.2°. The seven glucose units in the asymmetric unit are arranged in a left-handed helical fashion to form a U-shape. Distances from the center of the molecule to each O4 atom are in the range from 7.6 to 9.7 A indicating the presence of a large cavity in the molecule. Adjacent two glucose units in the asymmetric portion is in cis arrangement where the O2H hydroxyl group forms a hydrogen bond with O3H of next glucose unit. In contrast, symmetry-related glucose units are connected with trans arrangement and the O3H hydroxyl group is hydrogen-bonded to O6H of the adjacent glucose unit. The latter conformation ...

The role of alkyl chains in the stabilization of a bilayer structure of glycolipids in the crystalline state has been studied for a series of methyl 6-O-acyl-β-D-glucopyranosides and methyl 6-O-acyl-α-D-galactopyranosides. Thermal properties and crystal structures of these glycolipids with an odd number of alkyl carbon atoms in the acyl moiety have been investigated by thermal analysis and X-ray methods. The melting temperatures of methyl 6-O-acyl-β-D-glucopyranosides, which are lower than those of the corresponding methyl 6-O-acyl-α-D-galactopyranosides, decrease with increasing number of alkyl carbon atoms from one to seven, but increase for longer alkyl chains. The α-galactosides show only a monotonic decrease in melting temperature with increasing alkyl chain length. In the crystals, the molecules form a bilayer structure with interdigitated alkyl chains. The arrangement of sugar moieties in the crystals differs between the β-glucosides and the α-galactosides, but they are almost the same among sugar derivatives with various alkyl chain lengths. The packing of the acetyl moiety in a crystal of methyl 6-O-α-D-galactoside is different from that of the other α-galactoside derivatives with longer alkyl chains. The carbonyl oxygen atom causes steric hindrance with adjacent alkyl chains, and such unfavorable intermolecular contact explains the relatively low thermal stability of the crystal. On the other hand, the contribution of the β-glucoside to thermal stability is ascribed to the well-ordered packing of the long alkyl chain moiety.

The turkey-egg lysozyme (TEL) complex with tri-N-acetylchitotriose [(GlcNac)3] was co-crystallized from 1.5% TEL and 2 mM (GlcNac)3 at pH 4.2. The crystal structure was determined by molecular replacement and refined to an R value of 0.182 using 10-1.77 A data. The (GlcNac)3 molecule occupies the subsites A, B and C. At the subsites B and C, the sugar residues are bound in a similar manner to that found in the hen-egg lysozyme (HEL) complex. In contrast, the GlcNac residue at the subsite A is exposed to bulk solvent and has no contact with the protein molecule because the active residue Asp101 in HEL is replaced by Gly in TEL. A sulfate ion is bound in the vicinity of subsite B and forms hydrogen bonds with the sugar residue and the guanidino group of Arg61, assisting the binding of the sugar residue to subsite B. The active-site cleft of TEL is narrower than that of native TEL, thus attaining the best fit of the (GlcNac)3 molecule. The lack of binding ability of subsite A is discussed in relation to the catalytic properties of TEL. The result suggests that the cleavage pattern of oligosaccharide substrates in the catalytic reaction is regulated by the protein-sugar interaction at subsite A.

The importance of van der Waals contact between Glu 35 and Trp 109 to the active-site structure and the catalytic properties of human lysozyme (HL) has been investigated by site-directed mutagenesis. The X-ray analysis of mutant HLs revealed that both the replacement of Glu 35 by Asp or Ala, and the replacement of Trp 109 by Phe or Ala resulted in a significant but localized change in the active-site cleft geometry. A prominent movement of the backbone structure was detected in the region of residues 110 to 120 and in the region of residues 100 to 115 for the mutations concerning Glu 35 and Trp 109, respectively. Accompanied by the displacement of the main-chain atoms with a maximal deviation of C alpha atom position ranging from 0.7 A to 1.0 A, the mutant HLs showed a remarkable change in the catalytic properties against Micrococcus luteus cell substrate as compared with native HL. Although the replacement of Glu 35 by Ala completely abolished the lytic activity, HL-Asp 35 mutant retained a weak but a certain lytic activity, showing the possible involvement of the side-chain carboxylate group of Asp 35 in the catalytic action. The kinetic consequence derived from the replacement of Trp 109 by Phe or Ala together with the result of the structural change suggested that the structural detail of the cleft lobe composed of the residues 100 to 115 centered at Ala 108 was responsible for the turnover in the reaction of HL against the bacterial cell wall substrate. The results revealed that the van der Waals contact between Glu 35 and Trp 109 was an essential determinant in the catalytic action of HL.

Cyclodextrin glucanotransferase (CGTase) is an enzyme which produces cyclodextrins by the degradation of starch. The enzyme from alkalophilic Bacillus sp. 1011, consisting of 686 amino acid residues, was crystallized from the solution containing 20% PEG 3000 and 20% 2-propanol at pH 5.6 adjusted with citrate buffer. The space group was P1 and the unit cell contained two molecules (V(m) = 2.41 A(3) Da(-1)). The structure was solved by the molecular replacement method and refined to a conventional R value of 0.161 (R(free) = 0.211) for the reflections in the resolution range 1.8-10 A by energy minimization combined with simulated annealing. The molecule consists of five domains, designated A-E, and its backbone structure is similar to the structure of other bacterial CGTases. The molecule has two calcium binding sites where calcium ions are coordinated by seven ligands, forming a distorted pentagonal bipyramid. The two independent molecules are related by a pseudotwofold symmetry and are superimposed with an r.m.s. deviation value of 0.32 A for equivalent C(alpha) atoms. Comparison of these molecules indicated the relatively large mobility of domains C and E with respect to domain A. The active site is filled with water molecules forming a hydrogen-bond network with polar side-chain groups. Two water molecules commonly found in the active center of both molecules link to several catalytically important residues by hydrogen bonds and participate in maintaining a similar orientation of side chains in the two independent molecules.

The crystal structures and thermal properties of methyl 6-O-n-decanoyl-α-D-glucopyranoside (1), methyl 6-O-n-dodecanoyl-α-D-glucopyranoside (2), methyl 6-O-n-dodecanoyl-β-D-glucopyranoside (3), and methyl 6-O-n-dodecanoyl-α-D-galactopyranoside (4) were studied by X-ray and thermal analysis. Anhydrous crystals obtained from methanol solution by slow evaporation are monoclinic and in space group P2 1 with Z = 2. Cell dimensions : 1, a = 4.967(1) A, b = 7.513(1) A, c = 25.917(2) A, β = 92.92(1)° ; 2, a = 4.968(1) A, b = 7.503(1) A, c = 28.443(2) A, β = 92.68(1)° ; 3, a = 7.722(1) A, b = 7.346(1) A,c = 18.626(2) A, β = 91.51(1)° ; 4, a = 5.760(1) A, b = 7.986(1) A, c = 23.339(1) A, β = 90.21(1)°. The sugar moieties of 1 and 2 are disordered. The pyranose rings of β-D-glucopyranoside and α-D-galactopyranoside in 3 and 4, respectively, are in 4 C 1 chair conformation, and the alkyl chains in 1-4 are all-trans. Molecules are arranged in a bilayer structure with interdigitated alkyl chains. The hydrogen bonds between sugar moieties in 3 and 4, which are found only between adjacent layers, form infinite and finite chains in respective crystals. The melting point of compounds which have the same alkyl chain length are 70.1, 93.5, and 137.8°C for 2, 3, and 4, respectively. Epimers of these molecules exhibit packing arrangements which explain the wide variety in their melting temperatures.

In order to reveal the origin of carbohydrate recognition specificity of human lysozyme by clarifying the difference in the binding mode of ligands in the active site, the inactivation of human lysozyme by 2',3'-epoxypropyl beta-glycoside derivatives of the disaccharides, N,N'-diacetylchitobiose [GlcNAc-beta-(1-->4)-GlcNAc] and N-acetyllactosamine [Gal-beta-(1-->4)-GlcNAc], was investigated and the three-dimensional structures of the affinity-labeled enzymes were determined by X-ray crystallography at 1.7 A resolution. Under the conditions comprising 2.0 x 10(-3) M labeling reagent and 1.0 x 10(-5) M human lysozyme at pH 5.4, 37 degrees C, the reaction time required to reduce the lytic activity against Micrococcus luteus cells to 50% of its initial activity was lengthened by 3.7 times through the substitution of the nonreducing end sugar residue, GlcNAc to Gal. The refined structure of human lysozyme labeled by 2',3'-epoxypropyl beta-glycoside derivatives of N,N'-diacetylchitobiose (HL/NAG-NAG-EPO complex) indicated that the interaction mode of the N,N'-diacetylchitobiose moiety in substites B and C in this study was essentially the same as in the case of the complex of human lysozyme with the free ligand. On the other hand, the hydrogen-bonding pattern and the stacking interaction at subsite B were remarkably different between the HL/NAG-NAG-EPO complex and human lysozyme labeled by the 2',3'-epoxypropyl beta-glycoside of N-acetyllactosamine (HL/GAL-NAG-EPO complex). The reduced number of possible hydrogen bonds as well as the less favorable stacking between the side chain of Tyr63 in human lysozyme and the galactose residue in the HL/GAL-NAG-EPO complex reasonably explained the less efficient ability of the 2',3'-epoxypropyl beta-glycoside of N-acetyllactosamine as compared to that of N,N'-diacetylchitobiose as an affinity labeling reagent toward human lysozyme.

Wheat germ agglutinin isolectin 3 (WGA3) was crystallized from 10 mM acetate buffer at pH 4.9 containing 6 mM CaCl(2) and 4%(v/v) ethanol. The crystal belongs to monoclinic space group P2(1) with unit-cell dimensions a = 44.86, b = 91.02, c = 44.86 A, and beta = 110.22 degrees. The asymmetric unit contains two molecules (V(m) = 2.51 A(3) Da(-1)). The crystal structure was solved by the molecular-replacement method and was refined by the simulated-annealing method. The conventional R value was 0.191 for 19713 reflections [|F(o)| > 3sigma(F)] in the resolution range 8-1.9 A. The r.m.s. deviations from the ideal bond distances and angles were 0.014 A, and 3.0 degrees, respectively, and the estimated coordinate error was 0.2-0.25 A. The two molecules in the asymmetric unit are related by the pseudo twofold symmetry and form a dimer structure. The backbone structures of the two subunits are nearly identical with the r.m.s. difference of 0.36 A for the superposition of equivalent C(alpha) atoms. The dimer structure is very similar to those of isolectins 1 and 2 with the r.m.s. difference of 0.35-0.39 A for the C(alpha) superposition. Since amino-acid residues which differ from those of isolectin 1 or 2 are not involved in the contact between the two subunits, the subunit-subunit interaction is not significantly affected by the replacement of these residues. As a result, the geometry of the sugar-binding sites which are located at the interface between the two subunit molecules is basically conserved among three isolectins.

The crystal structures of methyl 6-O- n-octanoyl -α- d -galactopyranoside (1) and methyl 6-O- n-decanoyl -α- d -galactopyranoside (2) were investigated by X-ray analysis. Anhydrous crystals obtained from methanol solution by slow evaporation are monoclinic, and the space group is P21 with Z = 2. The cell dimensions are as follows, 1: a = 5.774(1), b = 8.013(1), c = 19.183(1) A , β = 98.50(1)°; 2 : a = 5.762(1), b = 8.003(1), c = 21.227(2) A , β = 93.93(1)° . The galactopyranoside ring is nearly in a 4C1 chair conformation but slightly distorted to twist boat and the alkyl chain is in all-trans conformation. In these crystals, molecules are arranged in a bilayer structure with interdigitated alkyl chains. The hydrogen bonding linkages between sugar moieties, which are found only between the layers, form an infinite chain through the crystal. 1 and 2 show higher melting points than those of corresponding β- d -glucopyranosides. The results indicate the important role of the sugar in the crystal.

Hexakis(2,6-di-O-methyl)-α-cyclodextrin (DMαCD) crystallizes in the space group P21 from 10% NaCl aqueous solution at room temperature and in the space group P212121 by the slow evaporation of water at ca. 90 °C. The P21 crystal shows a typical cage-type packing structure and a water molecule is included within its macrocyclic cavity. In the P212121 crystal, DMαCD molecules are arranged in a helically extended polymeric chain formed by the inclusion of an O-6CH3 methoxyl group of the adjacent molecule related by the twofold screw axis. In spite of the difference in the packing structure, the macrocyclic conformation of DMαCD molecules in the two crystals is nearly identical. The round structure of the macrocycle is maintained by the intramolecular O-3H—O-2 hydrogen bonds formed between adjacent residues. The rootmean-square distance between equivalent two atoms calculated after the least-squares superposition of the two molecules is 0.36 A for non-hydrogen atoms except O-6 and methyl carbon atoms.

The crystal structures of methyl 6-O-n- octanoyl -β- d - glucopyranoside 1 and methyl 6-O-n- decanoyl -β- d - glucopyranoside 2, which are types of glycosurfactants, were determined by X-ray analysis. Anhydrous crystals were obtained from a diethyl ether-acetone solution. The crystals are monoclinic, and the space group is P21 with Z = 2. The cell dimensions are as follows: 1, a = 7.760(1), b = 7.373(1), c = 15.514(1) A , β = 102.91(1)° ; 2, a = 7.724(1), b = 7.351(1), c = 16.957(2) A , β = 94.81(1)° . The glucopyranoside moieties are in a 4C1 chair conformation and the conformation of the alkyl chain is the all-trans type. The molecules are arranged parallel to the a–c plane in a bilayer structure where alkyl chains are interdigitated. The sugar moieties are stacked to form a hydrophilic zone elongated parallel to the ab plane and arranged with a packing structure similar to that of methyl β- d -glucopyranoside in spite of a substituted long alkyl chain. Hydrogen bondings of sugar moieties are found only between the bilayers.

Mono-2- O -methyl-, -2- O -ethyl-, and -2- O -allyl-cyclomaltoheptaose were prepared by alkylations of cyclomaltoheptaose in dilute aqueous alkali, and mono-2- O -propylcyclomaltoheptaose was obtained by hydrogenation of the allyl derivative. All the 2- O -alkyl derivatives were less soluble in water than was cyclomaltoheptaose. All formed inclusion complexes with toluene in aqueous solution, but only the methyl ether was less soluble in the water-toluene system than in water. The solubilities of the other ethers in water were enhanced by the addition of toluene. Partial methylation of cyclomaltoheptaose with 13 C-enriched dimethyl sulfate in dilute aqueous alkali yielded mixtures of products. The substitution patterns were analyzed by GLC-MS of the alditol acetates, prepared by hydrolysis, reduction, and acetylation, and by 13 C NMR after complete permethylation with nonenriched reagent. The results showed that methylation at O-2 is a predominant but not an exclusive reaction; as expected, the regioselectivity decreases with increasing degree of methylation.

A monoclinic crystal of hen egg lysozyme (HEL, E.C. 3.2.1.17) was obtained at 313 K from a 10%(w/v) NaCl solution at pH 7.6 containing 5%(v/v) 1-propanol. Cell dimensions were a = 27.23, b = 63.66, c = 59.12 A and beta = 92.9 degrees, and the space group was P2(1). The unit cell contains four molecules (V(m) = 1.79 A(3) Da(-1)). The structure was solved by the isomorphous replacement method with anomalous scattering followed by phase improvement by the solvent-flattening method. The refinement of the structure was carried out by the simulated-annealing method. The conventional R value was 0.187 for 18 260 reflections [|F(o)|3sigma(F)] in the resolution range 10-1.72 A. The r.m.s. deviations from the ideal bond distances and angles were 0.015 A and 3.0 degrees, respectively. The two molecules in the asymmetric unit are related by a translation of half a lattice unit along the a and c axes. The r.m.s. difference of equivalent C(alpha) atoms between the two molecules was 0.64 A and the largest difference was 3.57 A for Gly71. A significant structural change was observed in the regions of residues 45-50, 65-73 and 100-104. The residues 45-50, which connect two beta-strands, are shifted parallel to the beta-sheet plane between the two molecules. The residues 100-104 belong to the substrate-binding site (subsite A) and the high flexibility of this region may be responsible for the binding of the substrate and the release of reaction products.

A possibility of crystallography to estimate large conformational change of proteins is described. An example is the crystal structure of a mutant T4 lysozyme (Faber & Matthews, 1990), where four independent molecuels show a variety of hinge-bending angles. Another example is human α-lactalbumin (Harata & Muraki, 1992) . The amino acid residues of 104-110 assume α-helix in one crystal and a loop structure in another crystal. These observations demonstrate the large conformational flexibility of proteins and indicate that the X-ray crystallography is a powerful tool for the study of dynamic structure of proteins.

Mono-2- O -methyl-, -2- O -ethyl-, and -2- O -allyl-cyclomaltoheptaose were prepared by alkylations of cyclomaltoheptaose in dilute aqueous alkali, and mono-2- O -propylcyclomaltoheptaose was obtained by hydrogenation of the allyl derivative. All the 2- O -alkyl derivatives were less soluble in water than was cyclomaltoheptaose. All formed inclusion complexes with toluene in aqueous solution, but only the methyl ether was less soluble in the water-toluene system than in water. The solubilities of the other ethers in water were enhanced by the addition of toluene. Partial methylation of cyclomaltoheptaose with 13 C-enriched dimethyl sulfate in dilute aqueous alkali yielded mixtures of products. The substitution patterns were analyzed by GLC-MS of the alditol acetates, prepared by hydrolysis, reduction, and acetylation, and by 13 C NMR after complete permethylation with nonenriched reagent. The results showed that methylation at O-2 is a predominant but not an exclusive reaction; as expected, the regioselectivity decreases with increasing degree of methylation.

Crystal structure of hexakis(2,3,6-tri-O-acetyl)-α-cyclodextrin was determined by the X-ray method. The molecule with twofold crystallographic symmetry has a cavity with the shape of a rectangular box. Both ends of the cavity are closed by acetyl groups and a water molecules is included in the molecular cage.

The three-dimensional structure of a modified human lysozyme (HL), Glu 53 HL, in which Asp 53 was replaced by Glu, has been determined at 1.77 A resolution by X-ray analysis. The backbone structure of Glu 53 HL is essentially the same as the structure of wild-type HL. The root mean square difference for the superposition of equivalent C alpha atoms is 0.141 A. Except for the Glu 53 residue, the structure of the active site region is largely conserved between Glu 53 HL and wild-type HL. However, the hydrogen bond network differs because of the small shift or rotation of side chain groups. The carboxyl group of Glu 53 points to the carboxyl group of Glu 35 with a distance of 4.7 A between the nearest carboxyl oxygen atoms. A water molecule links these carboxyl groups by a hydrogen bond bridge. The active site structure explains well the fact that the binding ability for substrates does not significantly differ between Glu 53 HL and wild-type HL. On the other hand, the positional and orientational change of the carboxyl group of the residue 53 caused by the mutation is considered to be responsible for the low catalytic activity (ca. 1%) of Glu 53 HL. The requirement of precise positioning for the carboxyl group suggests the possibility that the Glu 53 residue contributes more than a simple electrostatic stabilization of the intermediate in the catalysis reaction.

The functional role of tyrosine-63 in the catalytic action of human lysozyme (EC 3.2.1.17) has been probed by site-directed mutagenesis. In order to identify the role of Tyr63 in the interaction with substrate, both the three-dimensional structures and the enzymatic functions of the mutants, in which Tyr63 was converted to phenylalanine, tryptophan, leucine, or alanine, have been characterized in comparison with those of the wild-type enzyme. X-ray crystallographical analysis of the mutant enzyme at not less than 1.77-A resolution indicated no remarkable change in tertiary structure except the side chain of 63rd residue. The conversion of Tyr63 to Phe or Trp did not change the enzymatic properties against the noncharged substrate (or substrate analogs) largely, while the conversion to Leu or Ala markedly reduced the catalytic activity to a few percent of wild-type enzyme. Kinetic analysis using p-nitrophenyl penta-N-acetyl-beta-(1----4)-chitopentaoside (PNP-(GlcNAc)5) as a substrate revealed that the reduction of activity should mainly be attributed to the reduction of affinity between enzyme and substrate. The apparent contribution of the phenolic hydroxyl group and the phenol group in the side chain of Tyr63 was estimated to 0.4 +/- 0.4 and 2.5 +/- 0.8 kcal mol-1, respectively. The result suggested that the direct contact between the planar side-chain group of Tyr63 and the sugar residue at subsite B is a major determinant of binding specificity toward a electrostatically neutral substrate in the catalytic action of human lysozyme.

The direct photoisomerization of (E)-4-(1-imidazoylmethyl)-cinnamic acid (IMC), a thromboxane synthetase inhibitor, to its (Z)-isomer at pH 2.0 was decelerated by beta-cyclodextrin (beta-CyD) and heptakis(2,6-di-O-methyl)-beta-cyclodextrin (DM-beta-CyD). The photostationary composition [(Z)-isomer:IMC ratio] was shifted in favor of IMC. These effects were much greater with DM-beta-CyD than with the parent beta-CyD. The quantum yield of the photoisomerization was significantly decreased by complex formation with beta-CyDs, whereas the extinction coefficient of the guest was only slightly decreased. This situation was in sharp contrast to those observed in less polar solvents and suggests that the suppressing mechanism with beta-CyD is different from that with less polar solvent systems. Spectroscopic studies (ultraviolet, circular dichroism, and nuclear magnetic resonance) indicated that IMC is tightly included in an axial mode in the cavity of DM-beta-CyD and that the rotation of the photoreactive site is sterically hindered. The results suggest that the suppressing effect of beta-CyDs on the photoisomerization of IMC results mainly from a steric origin.

A semi-empirical methodology is introduced for calculating the hydration enthalpy of organic molecules. The enthalpy term is decomposed into electrostatic and non-electrostatic contributions. The former is calculated on the basis of a reaction field theory developed in our previous studied. The theory is combined with the MNDO method. The non-electrostatic contributions are estimated using empirical equations derived from experimental data for some nonpolar molecules. For typical cation, anion and neutral molecules, an excellent correlation is found between the calculated and observed hydration enthalpies.