Development of a high throughput screening assay for inhibitors of fibroblast growth factor-receptor-heparin interactions.

High throughput screening (HTS) of large compound libraries for inhibitors of growth factors raises the requirement for simple yet reliable assays. Fibroblast growth factors (FGFs) play a pivotal role in the multistep pathway of malignant transformation, tumor progression, metastasis, and angiogenesis. FGF-2 (basic FGF) requires a cooperative interaction with heparin or heparan sulfate proteoglycans in order to form functional growth factor-receptor complexes that are essential for receptor binding and activation. We have developed a simple screening system, devised to identify molecules that modulate heparin-FGF-receptor interactions. The system is composed of a heparin matrix, FGF-2, and a FGF receptor-1 protein engineered by genetically fusing the extracellular domain of FGF receptor-1 to alkaline phosphatase (FRAP). The screen is conducted using 96-well plates to which heparin has been covalently attached. FGF-2 is then bound to the plates through heparin-FGF interactions, followed by the addition of FRAP and compounds to be screened for modulation of heparin-FGF, receptor-heparin, and receptor-FGF interactions. The endpoint of the assay is measured enzymatically using the alkaline phosphatase (AP)-catalyzed formation of a chromogenic product, which is directly proportional to the amount of FRAP present on the plates as a heparin-FGF-FRAP ternary complex. Reduced AP values relative to control, as measured by spectrophotometry, indicate inhibition of the formation of an active FGF-receptor-heparin complex. The simple and versatile nature of the assay makes it an attractive HTS system. The screen has identified several potent inhibitors of FGF-2 receptor binding and activation. Furthermore, secondary screening of the HTS-recognized compounds identified several compounds that have the capacity to block growth factor-mediated tumor progression and angiogenesis in vivo.

Cloning and functional expression of a human heparanase gene.

We have cloned a gene (HSE1) from a human placental cDNA library that encodes a novel protein exhibiting heparanase activity. The cDNA was identified through peptide sequences derived from purified heparanase isolated from human SK-HEP-1 hepatoma cells. HSE1 contains an open reading frame encoding a predicted polypeptide of 543 amino acids and possesses a putative signal sequence at its amino terminus. Northern blot analysis suggested strong expression of HSE1 in placenta and spleen. Transient transfection of HSE1 in COS7 cells resulted in the expression of a protein with an apparent molecular mass of 67-72 kDa. HSE1 protein was detectable in conditioned media but was also associated with the membrane fraction following cell lysis. The HSE1 gene product was shown to exhibit heparanase activity by specifically cleaving a labeled heparan sulfate substrate in a similar manner as purified native protein.

Properly oriented heparin-decasaccharide-induced dimers are the biologically active form of basic fibroblast growth factor.

Interaction of basic fibroblast growth factor (FGF-2) with heparin or heparan sulfate proteoglycans (HSPGs) is required for receptor activation and initiation of biological responses. To gain insight into the mechanism of activation of the FGF receptor by FGF-2 and heparin, we have used NMR, dynamic light scattering, and HSPG-deficient cells and cell-free systems. The first 28 N-terminal residues in FGF-2 were found to be highly mobile and flexible, consistent with the disorder found in both the NMR and X-ray structures. The structure of an FGF-2-heparin-decasaccharide complex that binds to and activates the FGF receptor was compared to a heparin-tetrasaccharide-induced complex that does not promote an interaction with the receptor. The major change observed upon the addition of the tetrasaccharide to FGF-2 was an increase in the correlation time consistent with the formation of an FGF-2 dimer. The NMR line widths of FGF-2 in the presence of the decasaccharide are severely broadened relative to the tetrasaccharide, consistent with dynamic light scattering results which indicate FGF-2 is a tetramer. The interaction of these heparin species with FGF-2 does not induce a significant conformational change in the overall structure of FGF-2, but small chemical shift changes are observed in both heparin and receptor binding sites. A trans-oriented symmetric dimer of FGF-2 is formed in the presence of the tetrasaccharide whereas two cis-oriented dimers in a symmetric tetramer are formed in the presence of the decasaccharide. This suggests that the cis-oriented FGF-2 dimer is the minimal biologically active structural unit of FGF-2. These data allow us to propose a novel mechanism to explain the functional interaction of FGF-2 with heparin and its transmembrane receptor.

High-resolution solution structure of basic fibroblast growth factor determined by multidimensional heteronuclear magnetic resonance spectroscopy.

The high-resolution solution structure of recombinant human basic fibroblast growth factor (FGF-2), a protein of 17.2 kDa that exhibits a variety of functions related to cell growth and differentiation, has been determined using three-dimensional heteronuclear NMR spectroscopy. A total of 30 structures were calculated by means of hybrid distance geometry--simulated annealing using a total of 2865 experimental NMR restraints, consisting of 2486 approximate inteproton distance restraints, 50 distance restraints for 25 backbone hydrogen bonds, and 329 torsion angle restraints. The atomic rms distribution about the mean coordinate positions for the 30 structures for residues 29-152 is 0.43 +/- 0.03 A for the backbone atoms, 0.83 +/- 0.05 A for all atoms, and 0.51 +/- 0.04 A for all atoms excluding disordered side chains. The overall structure of FGF-2 consists of 11 extended antiparallel beta-strands arranged in three groups of three or four strands connected by tight turns and loop regions creating a pseudo-3-fold symmetry. Two strands from each group come together to form a beta-sheet barrel of six antiparallel beta-strands. A helix-like structure was observed for residues 131-136, which is part of the heparin binding site (residues 128-138). The discovery of the helix-like region in the primary heparin binding site instead of the beta-strand conformation described in the X-ray structures may have important implications in understanding the nature of heparin--FGF-2 interactions. A total of seven tightly bound water molecules were found in the FGF-2 structure, two of which are located in the heparin binding site. The first 28 N-terminal residues appear to be disordered, which is consistent with previous X-ray structures. A best fit superposition of the NMR structure of FGF-2 with the 1.9 A resolution X-ray structure by Zhu et al. (1991) yields a backbone atomic rms difference of 0.94 A, indicative of a close similarity between the NMR and X-ray structures.

Flavins inhibit human cytomegalovirus UL80 protease via disulfide bond formation.

Among the most potent inhibitors of human cytomegalovirus protease identified by random screening of a chemical library was 1,4-dihydro-7,8-dimethyl 6H-pyrimido[1,2-b]-1,2,4,5-tetrazin-6-one (1) (PTH2). The oxidized form (2), PT, which is present in solutions of PTH2, was shown to be the actual inhibitory species which irreversibly inactivates the protease; recycling of PTH2 by dissolved oxygen results in complete inhibition of the protease at substoichiometric amounts of compound. No evidence for a covalent adduct between the protease and the inhibitor was obtained, and protease activity was restored by incubation of the inactivated enzyme with the reducing agent bismercaptoethyl sulfone, suggesting that disulfide bond formation was responsible for the observed inhibition. The five cysteines of the protease are normally in the reduced state; analysis of tryptic peptides from inhibited protease indicated that disulfide bonds Cys84-Cys87 and Cys138-Cys161 were formed. Using site-directed mutagenesis, the disulfide pair induced between Cys138 and Cys161 disulfide is dependent upon interaction of PT with the protease and does not form spontaneously, unlike that of the Cys84-Cys87 pair which can form in the absence of inhibitor. The inhibitor's redox chemistry is analogous to that of flavin, and, in fact, flavin inhibits the protease by the same mechanism, causing formation of a disulfide bond between Cys138 and Cys161. That the cysteines are dispensable, but can regulate protease activity by formation of a unique disulfide pair, suggests a plausible mechanism for control of proteolysis during the viral life cycle.

1H, 15N, 13C and 13CO assignments and secondary structure determination of basic fibroblast growth factor using 3D heteronuclear NMR spectroscopy.

The assignments of the 1H, 15N, 13CO and 13C resonances of recombinant human basic fibroblast growth factor (FGF-2), a protein comprising of 154 residues and with a molecular mass of 17.2 kDa, is presented based on a series of three-dimensional triple-resonance heteronuclear NMR experiments. These studies employ uniformly labeled 15N- and 15N-/13C-labeled FGF-2 with an isotope incorporation > 95% for the protein expressed in E. coli. The sequence-specific backbone assignments were based primarily on the interresidue correlation of C alpha, C beta and H alpha to the backbone amide 1H and 15N of the next residue in the CBCA(CO)NH and HBHA(CO)NH experiments and the intraresidue correlation of C alpha, C beta and H alpha to the backbone amide 1H and 15N in the CBCANH and HNHA experiments. In addition, C alpha and C beta chemical shift assignments were used to determine amino acid types. Sequential assignments were verified from carbonyl correlations observed in the HNCO and HCACO experiments and C alpha correlations from the HNCA experiment. Aliphatic side-chain spin systems were assigned primarily from H(CCO)NH and C(CO)NH experiments that correlate all the aliphatic 1H and 13C resonances of a given residue with the amide resonance of the next residue. Additional side-chain assignments were made from HCCH-COSY and HCCH-TOCSY experiments. The secondary structure of FGF-2 is based on NOE data involving the NH, H alpha and H beta protons as well as 3JHNH alpha coupling constants, amide exchange and 13C alpha and 13C beta secondary chemical shifts. It is shown that FGF-2 consists of 11 well-defined antiparallel beta-sheets (residues 30-34, 39-44, 48-53, 62-67, 71-76, 81-85, 91-94, 103-108, 113-118, 123-125 and 148-152) and a helix-like structure (residues 131-136), which are connected primarily by tight turns. This structure differs from the refined X-ray crystal structures of FGF-2, where residues 131-136 were defined as beta-strand XI. The discovery of the helix-like region in the primary heparin-binding site (residues 128-138) instead of the beta-strand conformation described in the X-ray structures may have important implications in understanding the nature of heparin-FGF-2 interactions. In addition, two distinct conformations exist in solution for the N-terminal residues 9-28. This is consistent with the X-ray structures of FGF-2, where the first 17-19 residues were ill defined.

Engineering of fibroblast growth factor: alteration of receptor binding specificity.

A five-residue loop structure in basic fibroblast growth factor (FGF-2) which extends from amino acid residue 118 to residue 122 was replaced, by cassette mutagenesis, with the corresponding seven-residue loop structure from the structural homologue acidic fibroblast growth factor (FGF-1) or the corresponding five-residue loop from interleukin-1 beta to give FGF-2LA and FGF-2LI, respectively. The mutants were expressed in Escherichia coli and purified to homogeneity, and their heparin and receptor binding and biological properties were examined. The ability of FGF-2LA to induce endothelial cell proliferation was the same as that of FGF-2. Affinities of the mutants to heparin and to cells that express FGF receptor-1 (FGFR-1) were identical to those of the wild-type protein. The role of the loop structure in FGF-1 and FGF-2 was elucidated by using soluble FGF receptor systems, which display distinct ligand binding specificities. Thus, FGF-2LA bound, with the same affinity as FGF-1 and FGF-2, to FGFR-1 and FGFR-2, whereas only FGF-1 and the FGF-1 loop-containing mutant, FGF-2LA, bound to the keratinocyte growth factor receptor. A change in receptor binding specificity was not observed with the FGF-2LI engineered mutant. That the binding specificity of FGF-2 was dramatically altered by transfer of a loop structure from FGF-1 to resemble the binding profile of the donor protein provides strong evidence that this motif is a receptor binding specificity determinant of fibroblast growth factors.

Diminished heparin binding of a basic fibroblast growth factor mutant is associated with reduced receptor binding, mitogenesis, plasminogen activator induction, and in vitro angiogenesis.

Using modeling of heparin-fibroblast growth factor interactions, we replaced four basic residues of basic fibroblast growth factor (FGF-2) with neutral glutamine residues by site-specific mutagenesis to give the mutants K128Q, K138Q, K128Q-K138Q, R129Q, K134Q, and R129Q-K134Q. The FGF mutants were characterized for their receptor and heparin binding affinities, mitogenic and cell proliferation activities, and their ability to induce plasminogen activator (PA) production and in vitro angiogenesis by cultured endothelial cells. Heparin binding properties and biological activities of the three mutants involving R129 and K134 remained essentially unchanged; however, significant changes for three mutants involving K128 and K138 were found. The KD values for heparin binding for K128Q and K138Q mutants were increased about 10-fold, and that for the K128Q-K138Q double mutant was increased by about 100-fold. The mutant K128Q-K138Q required a 10-fold higher concentration of heparin to promote binding to heparan sulfate proteoglycan (HSPG)-deficient CHO cells transfected with fibroblast growth factor receptor-1 (FGFR1) or to induce DNA synthesis in HSPG-deficient myeloid cells transfected with FGFR1. Binding affinities of the mutants to cell surface receptors on BHK-21 cells, however, were similar to that of wild-type FGF-2. In endothelial cell proliferation assays the activities of K128Q and K128Q-K138Q were about 10-fold lower than that of the wild-type protein, whereas the K138Q mutant exhibited wild-type activity. In addition, the K128Q-K138Q mutant displayed a markedly lowered capacity to induce PA activity in cultured endothelial cells and to form capillary-like structures in an in vitro angiogenesis model.(ABSTRACT TRUNCATED AT 250 WORDS)

Basic fibroblast growth factor protects endothelial cells against radiation-induced programmed cell death in vitro and in vivo.

Apoptosis (programmed cell death) serves as a common mechanism of interphase cell death after radiation exposure in thymic, lymphoid, and hematopoietic cells but has infrequently been documented in other adult mammalian cell types. The present study demonstrates that apoptotic interphase cell death occurs in endothelial cells after exposure to clinically relevant radiation doses and that basic fibroblast growth factor (bFGF) protects endothelial cells against this mode of the lethal effects of radiation. Radiation exposure produced heterologous double-stranded DNA breaks in endothelial cells, but the cells exhibited a similar competence for repair of this damage in the presence or absence of bFGF. However, subsequent to the completion of this repair process, a second process of DNA fragmentation became apparent, which was detected only in the absence of bFGF and was associated with a DNA ladder of oligonucleosomal fragments characteristic of apoptosis. The apoptotic DNA degradation occurred mainly in G0-G1 phase cells and was inhibited by bFGF stimulation. C3H/HeJ mice exposed to lethal doses of whole lung irradiation exhibited similar apoptotic changes in the endothelial cell lining of the pulmonary microvasculature within 6-8 h after radiation exposure. bFGF given i.v. immediately before and after irradiation inhibited the development of apoptosis in these cells and protected mice against the development of lethal radiation pneumonitis. These findings suggest that interphase apoptosis may represent a biologically relevant mechanism of radiation-induced cell kill in nonlymphoid mammalian cells both in vitro and in vivo and that natural protection mechanisms against this effect may be associated with the level of radiation resistance in normal and malignant tissues in vivo.

Refolding and characterization of human recombinant heparin-binding neurite-promoting factor.

Heparin-binding neurite-promoting factor (HBNF) is a highly basic, cysteine-rich 136-residue protein, and a member of a new class of heparin-binding proteins. It exhibits a neurite-outgrowth promoting activity and its expression is both temporally and spacially regulated during fetal and postnatal development. A high interspecies sequence conservation suggests important, presently unknown, biological functions. HBNF is structurally and most likely functionally related to the product of a developmentally regulated gene, MK (midkine). To elucidate biological roles of these proteins, recombinant forms of the proteins were produced. Expression of human recombinant HBNF and MK in Escherichia coli lead to the formation of insoluble aggregated protein that accounted for about 25% of the total cellular protein. Homogeneous, monomeric forms of each protein were recovered from inclusion bodies by reduction with dithiothreitol and solubilization in 8 M urea. Refolding of the reduced and denatured protein occurred upon dialysis at pH 7.4. Human recombinant (hr) HBNF and hrMK prepared in this manner were further purified by heparin affinity chromatography. Chromatographic evidence demonstrates that refolding and concomitant disulfide bond formation in hrHBNF proceeds in high yield with minimal formation of stable nonnative disulfides. Studies on the redox status of the 10 cysteine residues of bovine brain HBNF and the refolded recombinant protein indicate that all cysteines are engaged in disulfide bond formation. The disulfide arrangements for the recombinant protein were found to be identical to those in the native protein isolated from bovine brain.(ABSTRACT TRUNCATED AT 250 WORDS)

Fluorospectrometric analysis of heparin interaction with fibroblast growth factors.

The fluorescence emission of a single tryptophan residue present in both FGF-1 and FGF-2 was used as a structural probe to directly assess the interaction of the growth factors with heparin or beta-cyclodextran tetradecasulfate. About 20-25% of the fluorescence of either FGF-1 or FGF-2 is quenchable, and is dependent on sulfation of the ligands. The quenchable fluorescence is associated with about 20% of total FGF, suggesting the presence of two fluorospectrometric forms of the protein. The equilibrium dissociation constants, determined by this method, for heparin or beta-cyclodextrin tetradecasulfate binding to FGF-1 are about 1 nM, whereas the values for FGF-2 are 1 and 23 nM, respectively. The method provides a direct tool to evaluate FGF-ligand interaction and assess the structural integrity of the proteins.

Comparison of the disulfide bond arrangements of human recombinant and bovine brain heparin binding neurite-promoting factors.

Heparin binding neurite-promoting factor (HBNF) is a highly basic 136 amino acid protein containing 10 cysteine residues. We have determined the redox status and the disulfide arrangement of the cysteine residues in HBNF from bovine brain and refolded human recombinant protein produced in E. coli. Our data indicate that all 10 cysteines are involved in disulfide bond formation. The disulfide linkages of human recombinant and bovine brain HBNF, as determined after proteolytic digestions of the non-reduced proteins by peptide mapping and sequence analysis are: Cys15-Cys44, Cys23-Cys53, Cys30-Cys57, Cys67-Cys99 and Cys77-Cys109. Thus, recombinant HBNF has the same disulfide arrangement as the native brain-derived protein.

Preliminary X-ray crystallographic analysis of a modified basic fibroblast growth factor.

Human basic fibroblast growth factor (hbFGF) has been modified, with Ala3 and Ser5 substituted by glutamic acid, and the purified recombinant protein has been crystallized. The crystals are triclinic (space group P1) with unit cell parameters a = 31.0 A, b = 33.6 A, c = 34.7 A, alpha = 88 degrees, beta = 85 degrees, gamma = 76 degrees, and they diffract to at least 2 A.

Identification of a highly reactive threonine residue at the active site of gamma-glutamyl transpeptidase.

gamma-Glutamyl transpeptidase [(5-glutamyl)-peptide:amino-acid 5-glutamyltransferase, EC 2.3.2.2], an enzyme of major importance in glutathione metabolism, was inactivated by treating it with L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-[3-14C]isoxazoleacetic acid. This selective reagent binds stoichiometrically to the enzyme; more than 90% of the label was bound to its light subunit. Enzymatic digestion of the light subunit gave a 14C-labeled peptide that corresponds to amino acid residues 517-527 of the enzyme and two incomplete digestion products that contain this labeled peptide moiety. The radioactivity associated with this peptide was released with threonine-523 during sequencing by the automated gas-phase Edman method. The light subunit contains 14 other threonine residues and a total of 19 serine residues; these were not labeled. Threonine-523 is situated in the enzyme in an environment that greatly increases its reactivity, indicating that other amino acid residues of the enzyme must also participate in the active-site chemistry of the enzyme.

Activation of glutamate by gamma-glutamate kinase: formation of gamma-cis-cycloglutamyl phosphate, an analog of gamma-glutamyl phosphate.

gamma-Glutamate kinase, the enzyme that catalyzes the first step in the pathway from glutamate to proline, has been postulated to convert glutamate to a gamma-activated form (possibly gamma-glutamyl phosphate), which is reduced by a NADPH-linked reductase to yield glutamate gamma-semialdehyde (in equilibrium with delta 1-pyrroline-5-carboxylate). In the present work we found that the kinase, in the absence or presence of the reductase (and in the absence of NADPH), catalyzes stoichiometric formation of 5-oxo-L-proline and Pi from L-glutamate and ATP, but catalyzes hydroxamate formation at only about 10% of the rate of ATP-cleavage. A new substrate of the kinase was found; thus, cis-cycloglutamate (cis-1-amino-1,3-dicarboxycyclohexane), a glutamate analog which cannot cyclize to form an analog of 5-oxoproline, interacts effectively with the kinase. The trans form of cycloglutamate does not interact with the kinase; only the cis form can assume a diequatorial conformation equivalent to the extended conformation of glutamate. cis-Cycloglutamyl phosphate formation was shown and evidence was obtained for formation of an enzyme-ADP-cycloglutamyl phosphate complex. Although cis-cycloglutamyl phosphate is not a reducible substrate of the NADPH-dependent reductase, the findings indicate that it interacts with the reductase. These studies, which elucidate several aspects of the mechanism of the utilization of glutamate for formation of delta 1-pyrroline-5-carboxylate, support the hypothesis that the kinase and reductase function as an enzyme complex. A model is suggested in which gamma-glutamyl phosphate formed on the kinase interacts with the reductase to form a gamma-glutamyl-reductase complex, which is reduced by NADPH to yield glutamate gamma-semialdehyde.

Study of the 5-oxoprolinase reaction by 13C NMR.

5-Oxoprolinase catalyzes the ATP-dependent decyclization of 5-oxo-L-proline to L-glutamate. Previous studies provided evidence for the intermediate formation of a phosphorylated form of 5-oxoproline (Seddon, A. P., and Meister, A. (1986) J. Biol. Chem. 261, 11538-11541) and of a tetrahedral intermediate (Li, L., Seddon, A. P., and Meister, A. (1987) J. Biol. Chem. 262, 11020-11025). A new approach to the study of the reaction mechanism using the 18O isotope effect on the 13C NMR signals for 5-oxoproline and glutamate is reported here. The 13C chemical shifts induced by 18O substitution for the carbonyl group of 5-oxoproline and the gamma-carboxyl group of glutamate are about 0.03 ppm with respect to the corresponding 16O-compounds. Using 5-[18O]oxo[5-13C]proline (97 and 79.5 atom % excess, 13C and 18O, respectively), the disappearance of the 18O-labeled and unlabeled 5-oxoproline and formation of the corresponding glutamate species were followed in the reactions catalyzed by purified preparations of 5-oxoprolinase isolated from Pseudomonas putida and from rat kidney. This procedure permits simultaneous determinations of the rates of 18O exchange and of the overall decyclization reaction. The ratios of 18O exchange rates to the overall reaction rates for the bacterial and kidney enzyme catalyzed-reactions were 0.28 and 0.14, respectively. The findings support the view that the coupling of ATP hydrolysis to 5-oxoproline decyclization involves formation of a phosphorylated tetrahedal intermediate. Although the exchange phenomena are consistent with the mechanistic interpretations, they seem not to be required for catalysis.

Spermatogenic cell-somatic cell interactions are required for maintenance of spermatogenic cell glutathione.

Sertoli cells play a major role in the regulation of spermatogenic cell energy metabolism and differentiation. This study demonstrates that Sertoli cells are essential for the maintenance of spermatogenic cell glutathione (GSH), an important intracellular reductant and detoxicant. Primary spermatocytes and round spermatids isolated from Xenopus laevis contained 1.5 +/- 0.1 mM GSH, but sperm lacked detectable GSH. During a 5-day culture period, isolated spermatocytes and spermatids lost 80% of the initial GSH (t 1/2 = 55 h). The levels of GSH were unaffected by L-buthionine-SR-sulfoximine (BSO), a selective inhibitor of GSH synthesis. Cultures of testicular lobules and spermatocysts (composed of germ cells and Sertoli cells) depleted of interstitial tissue lost only 30% of their initial GSH in 4.5 days; the GSH levels decreased during treatment with BSO. Spermatogenic cells in cultured testes maintained their GSH levels for 7 days by a BSO-sensitive mechanism. These results demonstrate that the intracellular GSH levels of spermatogenic cells are dependent upon germ cell-somatic cell interactions. Spermatogenic cells were shown to possess gamma-glutamyl transpeptidase, glutathione synthetase, 5-oxoprolinase, and gamma-glutamylcysteine synthetase activities. [35S] Cysteine incorporation and distribution as analyzed by high performance liquid chromatography (HPLC) showed that isolated spermatogenic cells are capable of GSH synthesis. The rate of GSH synthesis, however, was insufficient to compensate for GSH turnover. These results demonstrate that production of spermatogenic cell GSH is dependent upon Sertoli cells. To our knowledge, this is the first evidence that interactions between different cell types may be of significance in GSH metabolism.

Interaction of the protein components of 5-oxoprolinase. Substrate-dependent enzyme complex formation.

5-Oxo-L-prolinase from Pseudomonas putida is composed of two reversibly dissociable proteins: Component A catalyzes 5-oxoproline-dependent cleavage of ATP, but does not catalyze the decyclization of 5-oxoproline; Component B is required for the coupling of ATP cleavage to ring-opening of 5-oxoproline to form glutamate (Seddon, A. P., Li, L., and Meister, A. (1984) J. Biol. Chem. 259, 8091-8094). We describe here the purifications of Components A and B to apparent homogeneity and the interactions between these two proteins. The cellular content of Component B activity is significantly greater than that of Component A. By gel filtration, Component A is a hexamer; but in the presence of substrates, it is a dimer. Component B can exist as an aggregate, an octamer, or a tetramer, depending upon the conditions used. Gel filtration of a mixture of Components A and B in the presence of substrates gives a unique protein species that exhibits 5-oxoprolinase activity. The Mr of this Component A-Component B complex indicates that it probably has an A2-B2 structure. The molar ratio of Component A to Component B in the complex was determined to be 1:1 by the continuous variation method (Job). Titrations of each component by the other suggest that phosphorylated 5-oxoproline-bound Component A is the entity that interacts with Component B. These studies indicate that the binding of phosphorylated 5-oxoproline-bound Component A to Component B to form a complex proceeds by a cooperative type mechanism. This is supported by the observed shifts of the intersection points of the Job curves (see Appendix).

18O studies on the 5-oxoprolinase reaction. Evidence for a phosphorylated tetrahedral intermediate.

5-Oxoprolinase catalyzes a reaction in which the cleavage of ATP to ADP and Pi and the decyclization of 5-oxoproline to form glutamate are coupled. When the reaction catalyzed by 5-oxoprolinase of Pseudomonas putida was carried out to 90% completion in H2(18)O, the residual 5-oxoproline was found to contain 18O in the amide carbonyl oxygen atom. Such isotopic incorporation was not observed in similar studies with a subunit of the enzyme which catalyzes 5-oxoproline-dependent ATPase and formation of a phosphorylated 5-oxoproline intermediate (Seddon, A.P., and Meister, A. (1986) J. Biol. Chem. 261, 11538-11543). When the complete reaction was carried out in H2(18)O, the products glutamate (gamma-carboxyl) and inorganic phosphate were mono- and di-labeled with 18O. Studies with 5-[18O]oxo-L-proline confirmed such replacement of the oxygen atoms of the gamma-carboxyl group of glutamate and the carbonyl oxygen of 5-oxoproline. Oxygen was not transferred from 5-oxoproline to inorganic phosphate. Studies with analogs of 5-oxoproline showed that di-labeling of inorganic phosphate occurred only when ATP hydrolysis was coupled or partially coupled with the decyclization of the substrate. Studies with 5-oxoprolinase from rat kidney gave similar results. These observations are in accord with the view that the reaction involves enzyme-bound phosphorylated intermediates and provide evidence for a phosphorylated tetrahedral intermediate, whose formation is required for coupling.

Trapping of an intermediate in the reaction catalyzed by 5-oxoprolinase.

Bacterial 5-oxoprolinase is composed of two protein components: Component A, which catalyzes 5-oxoproline-dependent ATP-hydrolysis and Component B, which couples the hydrolysis of ATP with the decyclization of 5-oxoproline to form glutamate (Seddon, A. P., Li, L., and Meister, A. (1984) J. Biol. Chem. 259, 8091-8094). Studies on this unusual enzyme system have led to evidence that an intermediate is formed by Component A. Application of the isotope-trapping method demonstrated an activated 5-oxoproline intermediate, whose formation requires ATP, Mg2+, and Component A. The amount of ATP-dependent trapping was close to the number of enzyme active sites. The intermediate formed by Component A was shown to be reducible by potassium borohydride to proline in low yield; when Component B was added, the formation of proline was abolished. Treatment of reaction mixtures containing Component A, 5-oxoproline, and [gamma-32P] ATP with diazomethane led to appearance of a 32P-labeled compound (found on thin layer chromatography), whose formation was significantly reduced when Component B was present. The new compound, which is labile, breaks down to form dimethyl[32P]phosphate. The total amount of dimethyl[32P]phosphate formed after breakdown is close to the number of active sites of Component A. The data are consistent with the conclusion that a phosphorylated form of 5-oxoproline is formed by Component A and suggest that Component B is required for conversion of this intermediate to glutamate.