Comparison of methods to estimate the affected body surface area and the dosage of topical treatments in psoriasis and atopic dermatitis: the advantage of a picture-based tool.

BACKGROUND: The accurate determination of the dosage of topical treatments is important given its repercussions on patient adherence and therapeutic efficacy. Up till now, the fingertip unit calculated by the rule of hands is considered the gold standard, although its use is associated with several drawbacks. OBJECTIVE: To compare different methods to estimate the affected body surface area (BSA) and dosage of topical treatments in atopic dermatitis and psoriasis and investigate its reliability, user-friendliness and timing. METHODS: In this study, we compared the reliability of three different methods: (i) the fingertip unit calculated by the 1% hand rule; (ii) a picture-based tool [termed Cutaneous Inflammatory Disease Extent Score (CIDES)]; and (iii) a digital drawing tool. Eleven observers scored 40 patients with psoriasis and eczema to assess the inter-rater and intrarater reliability. Timing was automatically recorded, and user-friendliness was investigated by a questionnaire. RESULTS: An excellent intraclass correlation (ICC) was found for both inter-rater agreement and intrarater agreement for the picture-based tool (ICC = 0.92 and ICC = 0.96, respectively). The ICCs for drawing the area of involvement on a silhouette were 0.89 and 0.93, respectively. Finally, the rule of hands was associated with an increased inter-rater variability although an excellent intrarater agreement was found (ICC = 0.79 and 0.95, respectively). Automated calculation of the amount of topical treatment improved reliability, and CIDES was associated with the least variation. CIDES was considered the preferred method by all observers and was fast to perform (median: 30 s). CONCLUSION: A picture-based method offered the most advantages (in terms of reliability, speed and user-friendliness) to estimate the affected BSA and calculate the dosage of topical treatments.

How do kinases transfer phosphoryl groups?

Understanding how phosphoryl transfer is accomplished by kinases, a ubiquitous group of enzymes, is central to many biochemical processes. Qualitative analysis of the crystal structures of enzyme-substrate complexes of kinases reveals structural features of these enzymes important to phosphoryl transfer. Recently determined crystal structures which mimic the transition state complex have added new insight into the debate as to whether kinases use associative or dissociative mechanisms of catalysis.

Crystallization of Ulex europaeus lectin I.

The lectin I from Ulex europaeus (UEAI) has a strong affinity for the H-type 2 human blood group determinant. Single crystals of UEAI have been grown in the monoclinic crystal system. Initial crystals were obtained after 11 months from a solution of 10 mg/ml protein, 40% 2,4-methylpentanediol and 0.1 N acetate buffer at pH 5.2. The technique of washing and reseeding was used to generate large suitable crystals. The space group is C2 with a = 78.84 A, b = 69.85 A, c = 120.62 A, beta = 108.74 degrees and Z = 4; there is one molecular dimer per asymmetric unit and the solvent content is estimated to be 58%. The crystals diffract to at least 2.8 A d spacings and are stable in the X-ray beam for more than three days.

The 1.8 A structure of the complex between chymostatin and Streptomyces griseus protease A. A model for serine protease catalytic tetrahedral intermediates.

The naturally occurring serine protease inhibitor, chymostatin, forms a hemiacetal adduct with the catalytic Ser195 residue of Streptomyces griseus protease A. Restrained parameter least-squares refinement of this complex to 1.8 A resolution has produced an R index of 0 X 123 for the 11,755 observed reflections. The refined distance of the carbonyl carbon atom of the aldehyde to O gamma of Ser195 is 1 X 62 A. Both the R and S configurations of the hemiacetal occur in equal populations, with the end result resembling the expected configuration for a covalent tetrahedral product intermediate of a true substrate. This study strengthens the concept that serine proteases stabilize a covalent, tetrahedrally co-ordinated species and elaborates those features of the enzyme responsible for this effect. We propose that a major driving force for the hydrolysis of peptide bonds by serine proteases is the non-planar distortion of the scissile bond by the enzyme, which thereby lowers the activation energy barrier to hydrolysis by eliminating the resonance stabilization energy of the peptide bond.

The 2.2 A resolution structure of the O(H) blood-group-specific lectin I from Ulex europaeus.

The tertiary and quaternary structure of the lectin I from Ulex europaeus (UE-I) has been determined to 2.2 A resolution. UE-I is a dimeric metalloglycoprotein that binds the H-type 2 human blood group determinant [alpha-L-Fucalpha(1-->2)-beta-D-Galbeta(1-->4)-beta-D-Glc NAcalpha-]. Nine changes from the published amino acid sequence were necessary to account for the electron density. The quaternary structural organization of UE-I is that of the most commonly occurring legume lectin dimer. The tertiary structure of the monomeric subunits is similar to that in the conventional lectin subunit; however, some structural differences are noted. These differences include a four-stranded anti-parallel "S" sheet in UE-I versus the five-stranded S sheet in other lectin monomers. The Ala residue of the Ala-Asp cis-peptide bond present in the carbohydrate-binding site of the conventional lectin monomer is replaced with a Thr in the UE-I structure. Also, a novel disulfide bridge linking Cys115 and Cys150 is present. There are two metallic ions, one calcium and the other manganese, per subunit. N-linked oligosaccharides are at residues 23 and 111 of each subunit. One molecule of R-2-methyl-2, 4-pentanediol (R-MPD) is present in a shallow depression on the surface of each subunit. In order to examine the binding of the H-type 2 blood group determinant by UE-I, its beta-methyl glycoside (H-type 2-OMe) was docked into the binding site of R-MPD. The epitope previously identified for H-type 2-OMe by chemical mapping proved, with only minor adjustment of amino acid residues, to be complementary to the shallow cavity occupied by R-MPD in the structure. Several key interactions have been proposed between the H-type 2-OMe and UE-I.

Preliminary crystallographic data for a monoclonal Fab fragment specific for HPr of the phosphoenolpyruvate: sugar phosphotransferase system of Escherichia coli.

Crystals for Fab fragments from a monoclonal antibody to HPr of the phosphoenopyruvate:sugar phosphotransferase system of Escherichia coli have been obtained from 14% polyethylene glycol 6000, 5 mM-Tris X HCl, 50 mM-sodium phosphate and 0.2 M-sodium chloride at pH 8.0. The space group is P2(1) with a = 110.85 A, b = 66.18 A, c = 67.21 A, beta = 113.0 degrees and Z = 4. The crystals exhibit the forms [100], [011] and [011] and the solvent content is 47%.

Crystallization of the calcium-activated phosphoenolpyruvate carboxykinase from Escherichia coli K12.

Single crystals of phosphoenolpyruvate carboxykinase from Escherichia coli K12 have been grown in the orthorhombic crystal system. Single crystals developed to a maximum size of 0.25 mm x 0.25 mm x 1.5 mm by the technique of washing and reseeding. The space group is P2(1)2(1)2(1), with a = 77.24 A, b = 89.18 A, c = 93.24 A and Z = 4; there is one enzyme molecule per crystallographic asymmetric unit and the solvent content is estimated to be 59%. The crystals diffract to at least 2.8 A d spacings and decompose in the X-ray beam after approximately two days of exposure.

Framework residues 71 and 93 of the chimeric B72.3 antibody are major determinants of the conformation of heavy-chain hypervariable loops.

Structural analysis derived from the crystallographic study of the chimeric B72.3 antibody illustrated that some heavy-chain framework residues having atomic interactions with heavy-chain CDR residues may directly affect the conformation of CDR loops. For example, an alanine residue at H71 provides room for packing CDR2/CDR1 and lysine residues at H73 and H93 contribute a salt-bridge to aspartic acid at H55 in CDR2 and a hydrogen bond to the carbonyl group at H96 in CDR3, respectively. We have analysed the contribution of these framework residues to the TAG72-binding affinity. We altered these framework residues by site-directed mutagenesis, and determined the affinity of these mutant chimeric antibodies for the TAG72 antigen by solid phase radioimmunoassay. We found that a single amino acid substitution of alanine by phenylalanine at H71 or lysine by isoleucine at H93, significantly reduced the binding affinity for the TAG72 antigen by 12 and 20-fold, respectively, whereas the substitution of lysine by alanine at H73 reduced the binding affinity only two-fold. Our results indicate that heavy-chain framework residues alanine at H71 and lysine at H93 of the chimeric B72.3 antibody are the major determinants of the conformation of heavy-chain CDR2/CDR1 and CDR3 loops, whereas the salt-bridge between lysine at H73 and aspartic acid at H55 is less important. The hydrogen bond between two framework residues, glutamine at H5 and serine at H25 does not affect any CDR conformation. Our results will thus be of importance especially when the humanized B72.3 antibody is constructed by grafting the CDR loops to a human framework. The important framework region interactions must be maintained in the final humanized antibody.

The phosphoryl-transfer mechanism of Escherichia coli phosphoenolpyruvate carboxykinase from the use of AlF(3).

The mechanism of reversible transfer of the gamma-phosphate group of ATP by Escherichia coli phosphoenolpyruvate carboxykinase (PCK) on to its substrate is of great interest. It is known that metallofluorides are accurate analogs of the transition state in the context of kinase mechanisms. Therefore, two complexes of PCK, one with AlF(3), Mg(2+) and ADP (complex I), the other with AlF(3), Mg(2+), ADP and pyruvate (complex II) were crystallized. The X-ray crystal structures of these two complexes were determined at 2.0 A resolution. The Al atom has trigonal bipyramidal geometry that mimics the transition state of phosphoryl transfer. The Al atom is at a distance of 2.8 A and 2.9 A from an oxygen atom of the beta-phosphoryl group of ADP in complex I and II, respectively. A water molecule in complex I and an oxygen atom of the pyruvate in complex II are located along the axis of the trigonal bipyramid on the side opposite to the beta-phosphoryl oxygen with respect to the equatorial plane, suggesting that the complexes are close mimics of the transition state. Along with the presence of positively charged species around the AlF(3) moiety, these results indicate that phosphoryl transfer occurs via a direct displacement mechanism with associative qualities.

The 2.5 A resolution structure of the jel42 Fab fragment/HPr complex.

The tertiary structure of Jel42 Fab fragment complexed with HPr, a phosphocarrier protein of the phosphoenolpyruvate:sugar phosphotransferase system of Escherichia coli, has been determined at 2.5 A resolution. X-ray diffraction from a larger crystal provided 22,067 unique reflections as compared to 14,763 unique reflections (2.8 A resolution), which were obtained previously from a smaller crystal. The higher resolution allowed for more precise location of amino acid side-chains and for the location of well-ordered water molecules. Five more residues in the Fab fragment are found to be involved in binding HPr and two additional residues are identified as part of the epitope, bringing the totals to 24 and 16, respectively. At least nine water molecules are found at the interface between the two proteins, and these mediate hydrogen bonding interactions between the Fab fragment and HPr. Three additional hydrogen bonds have been identified (bringing the total to ten) and one salt-bridge occurs between LysL50 of the L2 complementarity-determining region (CDR) and GluP66 of HPr. This salt-bridge is the only interaction between HPr and CDRL2; thus all six CDRs are involved in binding. Inspection and empirical energy minimization of mutant HPrs in the complex indicate that, in some cases in the binding interaction, water molecules may compensate for residue alterations. Binding to the mutant SerP64Tyr HPr may require a movement of the HPr main chain. The active centre region of HPr, which is not involved in binding the antibody, and which was not resolved in the 2.8 A resolution structure of the complex, was determined. This active centre determined at pH 5.8, which is completely free of intermolecular contacts due to crystal packing, shows a potential hydrogen bond between the AsnP12 OD1 atom and the HisP15 NE2 atom, and no involvement of the C terminus with HisP15. The HisP15 ND1 atom is the site of phosphorylation in HPr. Although a specific amino acid at residue 12 is not conserved in HPr molecules from all species, a hydrogen bond between the side-chains of residue 12 and HisP15 may be a conserved feature of the active centres.

Crystal structure of Escherichia coli phosphoenolpyruvate carboxykinase: a new structural family with the P-loop nucleoside triphosphate hydrolase fold.

The crystal structure of ATP-dependent phosphoenolpyruvate carboxykinase (ATP-oxaloacetate carboxy-lyase, (transphosphorylating), E.C. 4.1.1.49; PCK) from Escherichia coli strain K12 has been determined using a combination of multiple isomorphous replacement, density modification, and partial model phase combination, and refined to a conventional R-index of 0.204 (Rfree = 0.244) at 1.9 A resolution. Each PCK molecule consists of a 275 residue N-terminal domain and 265 residue C-terminal or mononucleotide-binding domain, with the active site postulated to be within a cleft between the two domains. PCK is an open-faced, mixed alpha/beta protein, with each domain having an alpha/beta folding topology as found in several other mononucleoside-binding enzymes. The putative phosphate-binding site of ATP adopts the P-loop motif common to many ATP and GTP-binding proteins, and is similar in structure to that found within adenylate kinase. However, the beta-sheet topology within the mononucleotide-binding fold of PCK differs from all other families within the P-loop containing nucleoside triphosphate hydrolase superfamily, therefore suggesting it represents the first member in a new family of such proteins. The mononucleotide-binding domain is also different in structure compared to the classical mononucleotide-binding fold (CMBF) common to adenylate kinase, p21ras, and elongation factor-Tu. Several amino acid residues, including R65, K212, K213, H232, K254, D269, K288 and R333 appear to make up the active site of the enzyme, and are found to be absolutely conserved among known members of the ATP-dependent PCK family. A cysteine residue is located near the active-site, as has been suggested for other PCKs, although in the E. coli enzyme C233 is buried and so is most likely not involved in substrate binding or catalysis. Two binding sites of the calcium-analog TB3+ have been determined, one within the active site coordinating to the side-chain of D269, and the other within the C-terminal domain coordinating to the side-chains of E508 and E511.

Refined structure of alpha-lytic protease at 1.7 A resolution. Analysis of hydrogen bonding and solvent structure.

The structure of alpha-lytic protease, a serine protease produced by the bacterium Lysobacter enzymogenes, has been refined at 1.7 A resolution. The conventional R-factor is 0.131 for the 14,996 reflections between 8 and 1.7 A resolution with I greater than or equal to 2 sigma (I). The model consists of 1391 protein atoms, two sulfate ions and 156 water molecules. The overall root-meansquare error is estimated to be about 0.14 A. The refined structure was compared with homologous enzymes alpha-chymotrypsin and Streptomyces griseus protease A and B. A new sequence numbering was derived based on the alignment of these structures. The comparison showed that the greatest structural homology is around the active site residues Asp102, His57 and Ser195, and that basic folding pathways are maintained despite chemical changes in the hydrophobic cores. The hydrogen bonds in the structure were tabulated and the distances and angles of interaction are similar to those found in small molecules. The analysis also revealed the presence of close intraresidue interactions. There are only a few direct intermolecular hydrogen bonds. Most intermolecular interactions involve bridging solvent molecules. The structural importance of hydrogen bonds involving the side-chain of Asx residues is discussed. All the negatively charged groups have a counterion nearby, while the excess positively charged groups are exposed to the solvent. One of the sulfate ions is located near the active site, whereas the other is close to the N terminus. Of the 156 water molecules, only seven are not involved in a hydrogen bond. Six of these have polar groups nearby, while the remaining one is in very weak density. There are nine internal water molecules, consisting of two monomers, two dimers and one trimer. No significant second shell of solvent is observed.

Preliminary X-ray data for the calmodulin/trifluoperazine complex.

Crystals of the calmodulin/trifluoperazine complex have been grown from 26% polyethylene glycol 4000 at pH 5.2 with 10 mM-calcium chloride, 10 mM-magnesium chloride and 1.2 mM-trifluoperazine at 14 degrees C. The crystals have space group P3(1)21 or P3(2)21 with a = 40.88 A, c = 180.9 A and Z = 6, and exhibit the forms [1010] and [0001]. There is one calmodulin molecule with two trifluoperazine molecules per asymmetric unit and the solvent content of the crystals is 51% (v/v).

The 1.6 A structure of histidine-containing phosphotransfer protein HPr from Streptococcus faecalis.

The histidine-containing phosphocarrier protein (HPr) is a central component of the phosphoenolpyruvate: sugar phosphotransferase system (PTS) that transports carbohydrates across the cell membrane of bacteria. The three-dimensional structure of Gram-positive Streptococcus faecalis HPr has been determined using the method of multiple isomorphous replacement. The R factor for all data is 0.156 for S. faecalis HPr at 1.6 A resolution with very good geometry. The overall folding topology of HPr is a classical open-faced beta-sandwich, consisting of four antiparallel beta-strands and three alpha-helices. Remarkable disallowed Ramachandran torsion angles of Ala16 at the active center, revealed by the X-ray structure of S. faecalis HPr, demonstrate a unique example of torsion-angle strain that is likely involved directly in protein function. A brief report concerning the torsion-angle strain has been presented recently. A newly-determined pH 7.0 structure is shown to have the same open conformation of the active center and the same torsion-angle strain at Ala16, suggesting that pH is not responsible for the structural observations. The current structure suggests a role for residues 12 and 51 in HPr's function, since they are involved in the active center through direct and indirect hydrogen-bonding interactions with the imidazole ring of His15. It is found that Ser46, the regulatory site in HPr from Gram-positive bacteria, N-caps the minor alpha-B helix and is also involved in the Asn43-Ser46 beta-turn. This finding, in conjunction with the proposed routes of communication between the regulatory site Ser46 and the active center in S. faecalis HPr, provides new insight into the understanding of how Ser46 might function. The putative involvement of the C-terminal alpha-carboxyl group and the related Gly67-Glu70 reverse beta-turn with respect to the function of HPr are described.

Structures of the lectin IV of Griffonia simplicifolia and its complex with the Lewis b human blood group determinant at 2.0 A resolution.

The structures of the fourth lectin isolated from Griffonia simplicifolia (GS4) and its complex with the methyl-glycoside of the Lewis b human blood group determinant (Le(b)-OMe) are reported at high resolution. The native GS4 crystal is isomorphous with the complexed GS4 crystal. The space group is P4(2)2(1)2 with unit cell dimensions a = 78.9 A, c = 89.1 A with one subunit of the lectin (bound to 1 Le(b)-OMe in the complex) in the crystallographic asymmetric unit. The native GS4 structure was solved by the molecular replacement technique and least-squares refined (PROLSQ and X-PLOR). The orientation of the Le(b)-OMe tetrasaccharide in the complex was established from a 2.8 A difference map with coefficients (Fcomplex--Fnative) and calculated phase angles from the native model. Both the final native and complex GS4 models consist of 1904 protein non-hydrogen atoms, one sulfate ion, one Ca ion, one Mn ion and three covalently-bound sugar residues N-linked to Asn18. In addition, the complex model has 47 Le(b)-OMe non-hydrogen atoms. The two structures have 135 water molecules in common in addition to eight and nine unique water molecules in the native and complex structures, respectively. The root-mean-square deviations from ideal bond distances and angles are 0.016 A, 3.2 degrees and 0.016 A, 3.0 degrees, for the native and complexed GS4, respectively. The R index for all unique data from 8 to 2.0 A is 0.187 for the native (19,204 reflections) and 0.181 for the complex (19,212 reflections). The tertiary structure of each subunit is similar to that of other leguminous lectins but the quaternary structure of the molecular dimer is different from that of any other lectin reported to date. The co-ordination about the Ca ion is pentagonal bipyramidal (with 1 long Ca(2+)-oxygen bond) and the co-ordination about the Mn ion is octahedral. Two conserved residues (Asp149 and Ser155) appear to be important because they are hydrogen-bonded to each other and to groups that co-ordinate the Mn ion. There are three cis-peptides in the polypeptide chain; two involve non-proline residues, one of which is homologous with other leguminous lectins and the other is unique to GS4. The two non-proline cis-peptides are located in the carbohydrate-binding site and are important for the specificity of the lectin. The molecular recognition of Le(b)-OMe by GS4 involves both polar and extensive non-polar interactions.(ABSTRACT TRUNCATED AT 400 WORDS)

The 1.9 A resolution structure of phospho-serine 46 HPr from Enterococcus faecalis.

The histidine-containing phosphocarrier protein HPr is a central component of the phosphoenolpyruvate:sugar phosphotransferase system (PTS), which transfers metabolic carbohydrates across the cell membrane in many bacterial species. In Gram-positive bacteria, phosphorylation of HPr at conserved serine 46 (P-Ser-HPr) plays several regulatory roles within the cell; the major regulatory effect of P-Ser-HPr is its inability to act as a phosphocarrier substrate in the enzyme I reaction of the PTS. In order to investigate the structural nature of HPr regulation by phosphorylation at Ser46, the structure of the P-Ser-HPr from the Gram- positive bacterium Enterococcus faecalis has been determined. X-ray diffraction analysis of P-Ser-HPr crystals provided 10,043 unique reflections, with a 95.1 % completeness of data to 1.9 A resolution. The structure was solved using molecular replacement, with two P-Ser-HPr molecules present in the asymmetric unit. The final R-value and R(Free) are 0.178 and 0.239, respectively. The overall tertiary structure of P-Ser-HPr is that of other HPr structures. However the active site in both P-Ser-HPr molecules was found to be in the "open" conformation. Ala16 of both molecules were observed to be in a state of torsional strain, similar to that seen in the structure of the native HPr from E. faecalis. Regulatory phosphorylation at Ser46 does not induce large structural changes to the HPr molecule. The B-helix was observed to be slightly lengthened as a result of Ser46 phosphorylation. Also, the water mediated Met51-His15 interaction is maintained, again similar to that of the native E. faecalis HPr. The major structural, and thus regulatory, effect of phosphorylation at Ser46 is disruption of the hydrophobic interactions between EI and HPr, in particular the electrostatic repulsion between the phosphoryl group on Ser46 and Glu84 of EI and the prevention of a potential interaction of Met48 with a hydrophobic pocket of EI.

M-DNA: A complex between divalent metal ions and DNA which behaves as a molecular wire.

M-DNA is a complex of DNA with divalent metal ions (Zn(2+), Co(2+), or Ni(2+)) which forms at pH conditions above 8. Upon addition of these metal ions to B-DNA at pH 8.5, the pH decreases such that one proton is released per base-pair per metal ion. Together with previous NMR data, this result demonstrated that the imino proton in each base-pair of the duplex was substituted by a metal ion and that M-DNA might possess unusual conductive properties. Duplexes of 20 base-pairs were constructed with fluorescein (donor) at one end and rhodamine (acceptor) at the other. Upon formation of M-DNA (with Zn(2+)) the fluorescence of the donor was 95 % quenched. Fluorescence lifetime measurements showed the presence of a very fast component in the decay kinetics with tau

Conversion of an antibody into an enzyme which cleaves the protein HPr.

Jel 42 is an IgG which binds to the small bacterial protein, HPr and the structure of the complex is known at high resolution. The IgG was expressed as a single chain variable fragment (scFv) and the binding to HPr was assessed by fluorescence polarization of fluorescein-labelled HPr. The binding constant for the IgG was about 20-fold higher than the scFv. Inspection of the structure of the complex suggested that it might be possible to convert the scFv into a bond-specific protease by the introduction of three catalytic residues: a glutamate to increase the nucleophilicity of a nearby water molecule, a lysine to increase the polarizability of the carbonyl group and a histidine to provide a proton to convert the amine into a better leaving group. By trial and error it was found that a fourth residue had to be converted into glycine in order to maintain the integrity of complimentarity-determining region three of the heavy chain (CDRH3) at the binding interface. The resulting quadruple mutant still bound to HPr and unlike other mutants, showed weak protease activity as judged from the fluorescence polarization assay. The activity was maximum at pH 6 consistent with a requirement for a protonated histidine residue. With the aid of HPr fluorescein-labelled at two different positions, it was demonstrated that the size of the products was consistent with cleavage occurring in the vicinity of the target peptide bond. The activity was specific for HPr since an excess of bovine serum albumin did not interfere with the reaction.

Complexes of aspartate aminotransferase with hydroxylamine derivatives: spectral studies in solution and in the crystalline state.

Hydroxylamine and its derivatives of general formula H2NOR react with aldehydes and aldimines to produce oximes. If R corresponds to the side chain of a natural amino acid, such compounds can be thought of as analogs of the corresponding amino acids, lacking the alpha-carboxylate group. Oximes formed between such compounds and pyridoxal phosphate in the active site of aspartate amino-transferase mimic external aldimine intermediates that occur during catalysis by this enzyme. The properties of oxime derivatives of mitochondrial aspartate aminotransferase with hydroxylamine and 6 compounds H2NOR were studied by absorption spectroscopy and circular dichroism in solution and by linear dichroism in crystals. Stable oximes, absorbing at lambda max congruent to 380 nm and exhibiting a negative Cotton effect, were obtained with the carboxylate-containing compounds. The oximes formed with carboxylate-free compounds showed somewhat different properties and stability. With H-Tyr a stable complex absorbing at lambda max congruent to 370 nm rather than at 380 nm, was obtained, H-Ala and H-Phe produced unstable oximes with the initial absorption band at lambda max congruent to 380 nm that was gradually replaced by a band at lambda max congruent to 340 nm. The species absorbing at 340 nm were shown to be coenzyme-inhibitor complexes which were gradually released from the enzyme. A similar 330-340 nm absorption band was observed upon reaction of the free coenzyme with all hydroxylamine inhibitors at neutral pH-values. The results of the circular dichroism experiments in solution and the linear dichroism studies in microcrystals of mAspAT indicate that the coenzyme conformation in these inhibitor/enzyme complexes is similar to that occurring in an external aldimine analogue, the 2-MeAsp/mAspAT complex. Co-crystallizations of the enzyme with the H2NOR compounds were also carried out. Triclinic crystals were obtained in all cases, suggesting that the "closed" structure cannot be stabilized by a single carboxylate group.