Establishment of ex vivo calibration curve for X-ray induced "dicentric + ring" and micronuclei in human peripheral lymphocytes for biodosimetry during radiological emergencies, and validation with dose blinded samples.

In the modern developing society, application of radiation has increased extensively. With significant improvement in the radiation protection practices, exposure to human could be minimized substantially, but cannot be avoided completely. Assessment of exposure is essential for regulatory decision and medical management as applicable. Until now, cytogenetic changes have served as surrogate marker of radiation exposure and have been extensively employed for biological dose estimation of various planned and unplanned exposures. Dicentric Chromosomal Aberration (DCA) is radiation specific and is considered as gold standard, micronucleus is not very specific to radiation and is considered as an alternative method for biodosimetry. In this study dose response curves were generated for X-ray induced "dicentric + ring" and micronuclei, in lymphocytes of three healthy volunteers [2 females (age 22, 23 years) and 1 male (24 year)]. The blood samples were irradiated with X-ray using LINAC (energy 6 MV, dose rate 6 Gy/min), in the dose range of 0-5Gy. Irradiated blood samples were cultured and processed to harvest metaphases, as per standard procedures recommended by International Atomic Energy Agency. Pooled data obtained from all the three volunteers, were in agreement with Poisson distribution for "dicentric + ring", however over dispersion was observed for micronuclei. Data ("dicentric + ring" and micronuclei) were fitted by linear quadratic model of the expression Y[bond, double bond]C + αD + ßD2 using Dose Estimate software, version 5.2. The data fit has resulted in linear coefficient α = 0.0006 (±0.0068) "dicentric + ring" cell-1 Gy-1 and quadratic coefficient ß = 0.0619 (±0.0043) "dicentric + ring" cell-1 Gy-2 for "dicentric + ring" and linear coefficient α = 0.0459 ± (0.0038) micronuclei cell-1 Gy-1 and quadratic coefficient ß = 0.0185 ± (0.0010) micronuclei cell-1 Gy-2 for micronuclei, respectively. Background frequencies for "dicentric + ring" and micronuclei were 0.0006 ± 0.0004 and 0.0077 ± 0.0012 cell-1, respectively. Established curves were validated, by reconstructing the doses of 8 dose blinded samples (4 by DCA and 4 by CBMN) using coefficients generated here. Estimated doses were within the variation of 0.9-16% for "dicentric + ring" and 21.7-31.2% for micronuclei respectively. These established curves have potential to be employed for biodosimetry of occupational, clinical and accidental exposures, for initial triage and medical management.

The comparison of Y chromosome microdeletion incidence in blood DNA and sperm cell DNA.

BACKGROUND: During gamete development and spermatogenesis, certain genes on the Y chromosome (Yq) in the Male-Specific Region (MSR) are responsible for human gametes formation. The long arm Yq is composed of both euchromatin and the genetically inactive heterochromatin regions. This region contains the Azoospermia factors AZFa, AZFb and AZFc. In the case of male infertility, microdeletions on the Yq chromosome appear to be structural chromosomal anomalies linked to sperm abnormality. METHODS: The present study aimed to look at the incidence, of Asthenospermia (AS), Teratospermia (TS), Oligospermia (OS) and Oligoasthenoteratospermia (OAT) patterns of Y chromosomal microdeletions in Indian infertile men with an (AZF a, b, c). This study was conducted with 75 infertile men as cases and 75 fertile men as a control for AZF locus microdeletion utilizing sequence-tagged sites. RESULTS: The AZFc region of germ cell DNA (50.6%) was the most deleted section in infertile men when compared to blood DNA (21.3%), followed by deletions in the AZFb region (21.3%) in germ cell DNA whereas blood DNA had no microdeletion in the AZFa region in both germ cell DNA and blood DNA. Infertile men displayed significant Yq microdeletion in both AZFb and also AZFc. Around 33% (25) of 75 infertile men had AZF (a, b, c) region microdeletion in blood DNA, compared to it germ cell DNA had a larger percentage of 72% (54) of Y chromosome microdeletions in the study samples. CONCLUSION: A high-frequency rate of microdeletions seen in germ cell DNA. PCR-based Y chromosome microdeletion screening using germ cell DNA along with Genomic DNA might help in screening for genetic abnormality in infertile men who endure assisted reproductive technology treatments. This study might be attributable to the interplay of lifestyle and genetic factors, both contributing to the risk of developing these germ-line deletions.

Crystal Structure Of Photorespiratory Alanine:Glyoxylate Aminotransferase 1 (AGT1) From Arabidopsis thaliana.

Photorespiration is an energetically costly metabolic pathway for the recycling of phosphoglycolate produced by the oxygenase activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (RUBISCO) to phosphoglycerate. Arabidopsis alanine:glyoxylate aminotransferase 1 (AGT1) is a peroxisomal aminotransferase with a central role in photorespiration. This enzyme catalyzes various aminotransferase reactions, including serine:glyoxylate, alanine:glyoxylate, and asparagine:glyoxylate transaminations. To better understand structural features that govern the specificity of this enzyme, its crystal structures in the native form (2.2-Å resolution) and in the presence of l-serine (2.1-Å resolution) were solved. The structures confirm that this enzyme is dimeric, in agreement with studies of the active enzyme in solution. In the crystal, another dimer related by noncrystallographic symmetry makes close interactions to form a tetramer mediated in part by an extra carboxyl-terminal helix conserved in plant homologs of AGT1. Pyridoxal 5'-phosphate (PLP) is bound at the active site but is not held in place by covalent interactions. Residues Tyr35' and Arg36', entering the active site from the other subunits in the dimer, mediate interactions between AGT and l-serine when used as a substrate. In comparison, AGT1 from humans and AGT1 from Anabaena lack these two residues and instead position a tyrosine ring into the binding site, which accounts for their preference for l-alanine instead of l-serine. The structure also rationalizes the phenotype of the sat mutant, Pro251 to Leu, which likely affects the dimer interface near the catalytic site. This structural model of AGT1 provides valuable new information about this protein that may enable improvements to the efficiency of photorespiration.

Crystal structures of the amino-terminal domain of LpoA from Escherichia coli and Haemophilus influenzae.

The bacterial periplasmic protein LpoA is an outer membrane lipoprotein and an activator for the cross-linking activity of PBP1A, a bifunctional peptidoglycan synthase. Previous structures of the amino-terminal (N) domain of LpoA showed it to consist entirely of helices and loops, with at least four tetratricopeptide-like repeats. Although the previously determined orthorhombic crystal structure of the N domain of Haemophilus influenzae LpoA showed a typical curved structure with a concave groove, an NMR structure of the same domain from Escherichia coli was relatively flat. Here, a crystal structure of the N domain of E. coli LpoA was determined to a resolution of 2.1 Šand was found to be more similar to the H. influenzae crystal structure than to the E. coli NMR structure. To provide a quantitative description for these comparisons, the various structures were superimposed pairwise by fitting the first half of each structure to its pairwise partner and then calculating the rotation axis that would optimally superimpose the second half. Differences in both the magnitude of the rotation and the direction of the rotation axis were observed between different pairs of structures. A 1.35 Šresolution structure of a monoclinic crystal form of the N domain of H. influenzae LpoA was also determined. In this structure, the subdomains rotate 10° relative to those in the original orthorhombic H. influenzae crystal structure to further narrow the groove between the subdomains. To accommodate this, a bound chloride ion (in place of sulfate) allowed the closer approach of a helix that forms one side of the groove.

Structural analyses of the Haemophilus influenzae peptidoglycan synthase activator LpoA suggest multiple conformations in solution.

In many Gram-negative bacteria, the peptidoglycan synthase PBP1A requires the outer membrane lipoprotein LpoA for constructing a functional peptidoglycan required for bacterial viability. Previously, we have shown that the C-terminal domain of Haemophilus influenzae LpoA (HiLpoA) has a highly conserved, putative substrate-binding cleft between two α/ß lobes. Here, we report a 2.0 Å resolution crystal structure of the HiLpoA N-terminal domain. Two subdomains contain tetratricopeptide-like motifs that form a concave groove, but their relative orientation differs by ∼45° from that observed in an NMR structure of the Escherichia coli LpoA N domain. We also determined three 2.0-2.8 Å resolution crystal structures containing four independent full-length HiLpoA molecules. In contrast to an elongated model previously suggested for E. coli LpoA, each HiLpoA formed a U-shaped structure with a different C-domain orientation. This resulted from both N-domain twisting and rotation of the C domain (up to 30°) at the end of the relatively immobile interdomain linker. Moreover, a previously predicted hinge between the lobes of the LpoA C domain exhibited variations of up to 12°. Small-angle X-ray scattering data revealed excellent agreement with a model calculated by normal mode analysis from one of the full-length HiLpoA molecules but even better agreement with an ensemble of this molecule and two of the partially extended normal mode analysis-predicted models. The different LpoA structures helped explain how an outer membrane-anchored LpoA can either withdraw from or extend toward the inner membrane-bound PBP1A through peptidoglycan gaps and hence regulate the synthesis of peptidoglycan necessary for bacterial viability.

Mean frequency and relative fluorescence intensity measurement of γ-H2AX foci dose response in PBL exposed to γ-irradiation: An inter- and intra-laboratory comparison and its relevance for radiation triage.

Measurement of γ-H2AX protein changes in the peripheral blood lymphocytes (PBL) of individuals exposed to ionizing radiation is a simple, sensitive, and rapid assay for radiation triage and early marker of dose estimation. The qualitative and quantitative measurements of the protein changes were examined using flow cytometry and microscopy. Whole blood and isolated lymphocytes were exposed in vitro between 0.1 and 5 Gy doses of (60) Co γ-radiation at a dose rate of 1 Gy/min. Radiation induced γ-H2AX foci frequency (n = 3) and relative fluorescence intensity (n = 7) in PBL was measured at 0.5 and 2 hrs postexposure. The observed dose response for γ-H2AX foci frequency at both time points, for whole blood and isolated lymphocytes did not show any significant (P > 0.05) differences. However, when compared with γ-H2AX foci frequency scored manually (microscopy), the semiautomated analysis (captured images) showed a better correlation (r(2) = 0.918) than that obtained with automated (Metafer) scoring (r(2) = 0.690). It is noteworthy to mention that, the γ-H2AX foci frequency quantified using microscopy showed a dose dependent increase up to 2 Gy and the relative fluorescence intensity (RFI) measured with flow cytometry revealed an increase up to 5 Gy in the PBL exposed in vitro. Moreover, a better correlation was observed between the γ-H2AX foci frequency obtained by manual scoring and RFI (r(2) = 0.910). Kinetic studies showed that the γ-H2AX foci remain more or less unchanged up to 4 hrs and reduces gradually over 48 hrs of postexposure at 37°C. Further, inter and intra-laboratory comparisons showed consistency in the scoring of γ-H2AX foci frequency by manual and semiautomated scoring. The overall results suggest that measurement of γ-H2AX (microscopy and flow cytometry) should be employed within 4 to 6 hrs for a reliable dosimetry either by sharing the work load between the laboratories or investing more manpower; however, triage can be possible even up to 48 hrs of postirradiation.

Delayed mitogenic stimulation decreases DNA damage assessed by micronucleus assay in human peripheral blood lymphocytes after (60)co irradiation.

While contradictory reports are available on the yield of dicentric chromosomes (DC) in blood samples stored at different temperature and stimulated to enter into cell cycle, various times gap followed by exposure, limited information is available on the micronucleus (MN) assay. As scoring the micronuclei frequency from the blood lymphocytes of exposed individuals is an alternative to the gold standard DC assay for triage applications, we examined radiation induced MN yield in delayed mitogenic stimulation after irradiation of in vitro. Peripheral blood lymphocytes (PBL) were exposed to low LET ((60)Co) radiation dose (0.1 to 5Gy) and incubated at 37°C for 2, 6 and 24 hours. The MN frequency obtained in blood samples stimulated 2 hours post-irradiation showed a dose dependent increase and used to construct the dose-response curve. Further, the results also showed that blood samples stimulated twenty four hours of post-irradiation, a significant reduction (p<0.05) in MN frequencies were obtained when compared to that of blood samples stimulated two hours and six hours after post-irradiation (0.5, 1, 3 and 5Gy). The observed result suggests that the prolonged PBL storage without mitogenic stimulation could lead to interphase cell death and a delayed blood sampling could results in underestimation of dose in biological dosimetry.

Dicentric chromosome aberration analysis using giemsa and centromere specific fluorescence in-situ hybridization for biological dosimetry: An inter- and intra-laboratory comparison in Indian laboratories.

To facilitate efficient handling of large samples, an attempt towards networking of laboratories in India for biological dosimetry was carried out. Human peripheral blood samples were exposed to (60)Co γ-radiation for ten different doses (0-5Gy) at a dose rate of 0.7 and 2Gy/min. The chromosomal aberrations (CA) were scored in Giemsa-stained and fluorescence in-situ hybridization with centromere-specific probes. No significant difference (p>0.05) was observed in the CA yield for given doses except 4 and 5Gy, between the laboratories, among the scorers and also staining methods adapted suggest the reliability and validates the inter-lab comparisons exercise for triage applications.

Structure and characterization of the 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase from Aeropyrum pernix.

The first enzyme in the shikimic acid biosynthetic pathway, 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7PS), varies significantly in size and complexity in the bacteria and plants that express it. The DAH7PS from the archaebacterium Aeropyrum pernix (DAH7PS(Ap)) is among the smallest and least complex of the DAH7PS enzymes, leading to the hypothesis that DAH7PS(Ap) would not be subject to feedback regulation by shikimic acid pathway products. We overexpressed DAH7PS(Ap) in Escherichia coli, purified it, and characterized its enzymatic activity. We then solved its X-ray crystal structure with a divalent manganese ion and phosphoenolpyruvate bound (PDB ID: 1VS1). DAH7PS(Ap) is a homodimeric metalloenzyme in solution. Its enzymatic activity increases dramatically above 60 °C, with optimum activity at 95 °C. Its pH optimum at 60 °C is 5.7. DAH7PS(Ap) follows Michaelis-Menten kinetics at 60 °C, with a K(M) for erythrose 4-phosphate of 280 µM, a K(M) for phosphoenolpyruvate of 891 µM, and a k(cat) of 1.0 s(-1). None of the downstream products of the shikimate biosynthetic pathway we tested inhibited the activity of DAH7PS(Ap). The structure of DAH7PS(Ap) is similar to the structures of DAH7PS from Thermatoga maritima (PDB ID: 3PG8) and Pyrococcus furiosus (PDB ID: 1ZCO), and is consistent with its designation as an unregulated DAH7PS.

Structure of YraM, a protein essential for growth of Haemophilus influenzae.

Nontypeable Haemophilus influenzae is an obligate human parasite that often causes middle ear infections in children and exacerbates chronic obstructive pulmonary disorder, the fourth leading cause of death in the United States. There are no effective vaccines available for this strain. The lipoprotein YraM (gene HI1655) was identified as essential for the growth and viability of H. influenzae but its function is unknown. Sequence comparisons showed that YraM is a fusion of two protein modules. We grew crystals of the carboxyl-terminal module of YraM comprising residues 257-573 (YraM-C), phased the diffraction data by the multiwavelength anomalous diffraction technique, and refined the model to a crystallographic R-factor of 0.16 (R(free) = 0.19) with data to 1.35 A resolution. The two-domain structure of YraM-C adopts a fold similar to that observed for the open, unliganded forms of several periplasmic binding proteins (PBPs) involved in bacterial active transport. Sequence alignments of YraM homologues from other Gram-negative species showed that the most conserved residues of YraM-C cluster between the two domains in the location where other PBPs bind their cognate ligand. Modeling of YraM-C into a closed conformation similar to the leucine-bound form of the Leu/Ile/Val-binding protein (LIVBP) shows a putative binding pocket larger than the leucine-binding site in LIVBP. The pocket has both polar and nonpolar surfaces, with the latter located in the same area where a leucine side chain binds to LIVBP. We discuss possible biological functions of YraM considering its predicted location in the outer membrane, a novel place for such a binding protein.

The 2.2 A crystal structure of Hsp33: a heat shock protein with redox-regulated chaperone activity.

BACKGROUND: One strategy that cells employ to respond to environmental stresses (temperature, oxidation, and pathogens) is to increase the expression of heat shock proteins necessary to maintain viability. Several heat shock proteins function as molecular chaperones by binding unfolded polypeptides and preventing their irreversible aggregation. Hsp33, a highly conserved bacterial heat shock protein, is a redox-regulated molecular chaperone that appears to protect cells against the lethal effects of oxidative stress. RESULTS: The 2.2 A crystal structure of a truncated E. coli Hsp33 (residues 1-255) reveals a domain-swapped dimer. The core domain of each monomer (1-178) folds with a central helix that is sandwiched between two beta sheets. The carboxyl-terminal region (179-235), which lacks the intact Zn binding domain of Hsp33, folds into three helices that pack on the other subunit. The interface between the two core domains is comprised of conserved residues, including a rare Glu-Glu hydrogen bond across the dyad axis. Two potential polypeptide binding sites that span the dimer are observed: a long groove containing pockets of conserved and hydrophobic residues, and an intersubunit 10-stranded beta sheet "saddle" with a largely uncharged or hydrophobic surface. CONCLUSIONS: Hsp33 is a dimer in the crystal structure. Solution studies confirmed that this dimer reflects the structural changes that occur upon activation of Hsp33 as a molecular chaperone. Patterns of conserved residues and surface charges suggest that two grooves might be potential binding sites for protein folding intermediates.

Activation of the redox-regulated molecular chaperone Hsp33--a two-step mechanism.

BACKGROUND: Hsp33 is a novel redox-regulated molecular chaperone. Hsp33 is present in the reducing environment of the cytosol and is, under normal conditions, inactive. The four highly conserved cysteines found in Hsp33 constitute a novel zinc binding motif. Upon exposure to oxidative stress, Hsp33's chaperone activity is turned on. This activation process is initiated by the formation of two intramolecular disulfide bonds. Recently, the 2.2 A crystal structure of Hsp33 has been solved, revealing that Hsp33 is present as a dimer in the structure (Vijayalakshmi et al., this issue, 367-375 [1]). RESULTS: We show here that oxidized, highly active Hsp33 is a dimer in solution. In contrast, reduced and inactive Hsp33 is monomeric. The incubation of reduced Hsp33 in H(2)O(2) leads to the simultaneous formation of two intramolecular disulfide bonds and the concomitant release of zinc. This concentration-independent step is followed by a concentration-dependent association reaction. The dimerization of Hsp33 requires highly temperature-sensitive structural rearrangements. This allows Hsp33's activation process to be greatly accelerated at heat shock temperatures. CONCLUSIONS: The regulation of Hsp33's chaperone function is highly sophisticated. On a transcriptional level, Hsp33 is under heat shock control. This increases the concentration of Hsp33 under heat and oxidative stress, a process that favors dimerization, a critical step in Hsp33's activation reaction. On a posttranslational level, Hsp33 is redox regulated. Dimerization of disulfide-bonded Hsp33 monomers leads to the formation of two extended, putative substrate binding sites. These sites might explain Hsp33's high and promiscuous affinity for unstructured protein folding intermediates.

Visualization of intermediate and transition-state structures in protein-tyrosine phosphatase catalysis.

Engineering site-specific amino acid substitutions into the protein-tyrosine phosphatase (PTPase) PTP1 and the dual-specific vaccinia H1-related phosphatase (VHR), has kinetically isolated the two chemical steps of the reaction and provided a rare opportunity for examining transition states and directly observing the phosphoenzyme intermediate. Changing serine to alanine in the active-site sequence motif HCXXGXXRS shifted the rate-limiting step from intermediate formation to intermediate hydrolysis. Using phosphorus 31P NMR, the covalent thiol-phosphate intermediate was directly observed during catalytic turnover. The importance of the conserved aspartic acid (D92 in VHR and D181 in PTP1) in both chemical steps was established. Kinetic analysis of D92N and D181N mutants indicated that aspartic acid acts as a general acid by protonating the leaving-group phenolic oxygen. Structure-reactivity experiments with native and aspartate mutant enzymes established that proton transfer is concomitant with P-O cleavage, such that no charge develops on the phenolic oxygen. Steady- and presteady-state kinetics, as well as NMR analysis of the double mutant D92N/S131A (VHR), suggested that the conserved aspartic acid functions as a general base during intermediate hydrolysis. As a general base, aspartate would activate a water molecule to facilitate nucleophilic attack. The amino acids involved in transition-state stabilization for cysteinylphosphate hydrolysis were confirmed by the x-ray structure of the Yersinia PTPase complexed with vanadate, a transition-state mimic that binds covalently to the active-site cysteine. Consistent with the NMR, x-ray, biochemical, and kinetic data, a unifying mechanism for catalysis is proposed.

The isomorphous structures of prethrombin2, hirugen-, and PPACK-thrombin: changes accompanying activation and exosite binding to thrombin.

The X-ray crystal structure of prethrombin2 (pre2), the immediate inactive precursor of alpha-thrombin, has been determined at 2.0 A resolution complexed with hirugen. The structure has been refined to a final R-value of 0.169 using 14,211 observed reflections in the resolution range 8.0-2.0 A. A total of 202 water molecules have also been located in the structure. Comparison with the hirugen-thrombin complex showed that, apart from the flexible beginning and terminal regions of the molecule, there are 4 polypeptide segments in pre2 differing in conformation from the active enzyme (Pro 186-Asp 194, Gly 216-Gly 223, Gly 142-Pro 152, and the Arg 15-Ile 16 cleavage region). The formation of the Ile 16-Asp 194 ion pair and the specificity pocket are characteristic of serine protease activation with the conformation of the catalytic triad being conserved. With the determination of isomorphous structures of hirugen-thrombin and D-Phe-Pro-Arg chloromethyl ketone (PPACK)-thrombin, the changes that occur in the active site that affect the kinetics of chromogenic substrate hydrolysis on binding to the fibrinogen recognition exosite have been determined. The backbone of the Ala 190-Gly 197 segment in the active site has an average RMS difference of 0.55 A between the 2 structures (about 3.7 sigma compared to the bulk structure). This segment has 2 type II beta-bends, the first bend showing the largest shift due to hirugen binding. Another important feature was the 2 different conformations of the side chain of Glu 192. The side chain extends to solvent in hirugen-thrombin, which is compatible with the binding of substrates having an acidic residue in the P3 position (protein-C, thrombin platelet receptor). In PPACK-thrombin, the side chain of Asp 189 and the segment Arg 221A-Gly 223 move to provide space for the inhibitor, whereas in hirugen-thrombin, the Ala 190-Gly 197 movement expands the active site region. Although 8 water molecules are expelled from the active site with PPACK binding, the inhibitor complex is resolvated with 5 other water molecules.

Effect of the 7-amino substituent on the inhibitory potency of mechanism-based isocoumarin inhibitors for porcine pancreatic and human neutrophil elastases: a 1.85-A X-ray structure of the complex between porcine pancreatic elastase and 7-[(N-tosylphenylalanyl)amino]-4-chloro-3- methoxyisocoumarin.

A series of new acyl, urea, and carbonate derivatives of 7-amino-4-chloro-3-methoxyisocoumarin were synthesized and evaluated as irreversible inhibitors of human neutrophil elastase (HNE) and porcine pancreatic elastase (PPE). Inhibition of HNE is directly related to the hydrophobicity of the substituent on the 7-amino group. The N-Tos-Phe derivative (19) is the best HNE inhibitor with a second-order rate constant kobs/[I] = 200,000 M-1 s-1. The closest analogue in this series, the 3,3-diphenylpropionyl derivative 5, had a kobs/[I] = 130,000 M-1 s-1 with HNE. In contrast to the Tos-Phe derivative 19, phenylacetyl derivative 2 and carbonates 22 and 25 gave extremely stable enzyme-inhibitor complexes with deacylation half-lives longer than 48 h with both elastases. N-Phenylurea derivative 25 was the best inhibitor for PPE with a second-order rate constant kobs/[I] = 7300 M-1 s-1. The crystal structure of a complex of PPE with N-tosyl-Phe derivative 19 was determined at 1.85-A resolution and refined to a final R factor of 16.9%. The isocoumarin forms an acyl enzyme with Ser-195, while His-57 is near the inhibitor, but not covalently linked. The Tos-Phe makes a few hydrophobic contacts with the S' subsites of PPE, but appears to be interacting primarily with itself in the PPE structure. This region of HNE is more hydrophobic and modeling indicates that the inhibitor would probably make additional contacts with the enzyme.

Structural study of porcine pancreatic elastase complexed with 7-amino-3-(2-bromoethoxy)-4-chloroisocoumarin as a nonreactivatable doubly covalent enzyme-inhibitor complex.

The complex of porcine pancreatic elastase (PPE) with 7-amino-3-(2-bromoethoxy)-4-chloroisocoumarin, a potent mechanism-based inhibitor, was crystallized and the crystal structure determined at 1.9-A resolution with a final R factor of 17.1%. The unbiased difference Fourier electron density map showed continuous density from O gamma of Ser 195 to the benzoyl carbonyl carbon atom and from N epsilon 2 of His 57 to the carbon atom at the 4-position of the isocoumarin ring in the inhibitor. This suggested unambiguously that the inhibitor was doubly covalently bound to the enzyme. It represents the first structural evidence for irreversible binding of an isocoumarin inhibitor to PPE through both Ser 195 and His 57 in the active site. The PPE-inhibitor complex is only partially activated in solution by hydroxylamine and confirms the existence of the doubly covalently bound complex along with the acyl enzyme. The benzoyl carbonyl oxygen atom of the inhibitor is not situated in the oxyanion hole formed by the amide (greater than NH) groups of Gly 193 and Ser 195. The complex is stabilized by the hydrogen-bonding interactions in the active site (from the N epsilon 2 of Gln 192 to the bromine atom in the inhibitor and the amino group at the 7-position of the isocoumarin ring to the carbonyl oxygen of Thr 41) and by van der Waals interactions. The inhibition rates of several 7-substituted 4-chloro-3-(bromoalkoxy)isocoumarins toward PPE were measured.(ABSTRACT TRUNCATED AT 250 WORDS)