Using deterministic record linkage to link ambulance and emergency department data: is it possible without patient identifiers? A case study from the UK.

INTRODUCTION: Routine linkage of emergency ambulance records with those from the emergency department is uncommon in the UK. Our study, known as the Pre-Hospital Emergency Department Data Linking Project (PHED Data), aimed to link records of all patients conveyed by a single emergency ambulance service to thirteen emergency departments in the UK from 2012-2016. OBJECTIVES: We aimed to examine the feasibility and resource requirements of collecting de-identified emergency department patient record data and, using a deterministic matching algorithm, linking it to ambulance service data. METHODS: We used a learning log to record contacts and activities undertaken by the research team to achieve data linkage. We also conducted semi-structured interviews with information management/governance staff involved in the process. RESULTS: We found that five steps were required for successful data linkage for each hospital trust. The total time taken to achieve linkage was a mean of 65 weeks. A total of 958,057 emergency department records were obtained and, of these, 81% were linked to a corresponding ambulance record. The match rate varied between hospital trusts (50%-94%). Staff expressed strong enthusiasm for data linkage. Barriers to successful linkage were mainly due to inconsistencies between and within acute trusts in the recording of two ambulance event identifiers (CAD and call sign). Further data cleaning was required on emergency department fields before full analysis could be conducted. Ensuring the data was not re-identifiable limited validation of the matching method. CONCLUSION: We conclude that deterministic record linkage based on the combination of two event identifiers (CAD and call sign) is possible. There is an appetite for data linkage in healthcare organisations but it is a slow process. Developments in standardising the recording of emergency department data are likely to improve the quality of the resultant linked dataset. This would further increase its value for providing evidence to support improvements in health care delivery. HIGHLIGHTS: Ambulance records are rarely linked to other datasets; this study looks at the feasibility and resource requirement to use deterministic matching to link ambulance and emergency department data for patients conveyed by ambulance to the emergency department.It is possible to link these data, with an average match rate of 81% across 13 emergency departments and one large ambulance trust.All trusts approached provided match-able data and there was an appetite for data linkage; however, it was a long process taking an average of 65 weeks.We conclude that deterministic matching using no patient identifiers can be used in this setting.

Patch testing over tattoos.

BACKGROUND: There is little, if any, literature regarding placing and reading of patch-tests on tattooed skin. METHOD: A patient whose entire back was covered with multiple tattoos was patch-tested. RESULTS: Multiple positive patch-tests were seen with no apparent reduction in intensity related to tattooed skin. CONCLUSION: If necessary, patch-tests can be placed and read on tattooed skin.

Mechanistic effects of autophosphorylation on receptor tyrosine kinase catalysis: enzymatic characterization of Tie2 and phospho-Tie2.

Activation of receptor tyrosine kinases by autophosphorylation is one of the most common and critical transformations in signal transduction, yet its role in catalysis remains controversial. Autophosphorylation of the angiogenic receptor tyrosine kinase Tie2 was studied in terms of the autophosphorylation sites, sequence of phosphorylation at these sites, kinetic effects, and mechanistic consequences. Isoelectric focusing electrophoresis and mass spectrometric analysis of a Tie2 autophosphorylation time course showed that Tyr992 on the putative activation loop was phosphorylated first followed by Tyr1108 in the C-terminal tail (previously unidentified autophosphorylation site). Autophosphorylation of Tie2 to produce pTie2 resulted in a 100-fold increase in k(cat) and a 460-fold increase in k(cat)/K(m). Viscosity studies showed that the unphosphorylated Tie2 was partially limited by product diffusion ((k(cat))(eta) = 0.67 +/- 0.06), while product release was more rate-limiting ((k(cat))(eta) = 0.94 +/- 0.08) for autophosphorylated Tie2 (pTie2). Furthermore, autophosphorylation did not significantly affect the phosphoacceptor dissociation constants. There was a significant (k(cat))(H)/(k(cat))(D) solvent isotope effect (SIE) for unphosphorylated Tie2 (2.42 +/- 0.12) and modest SIE (1.28 +/- 0.04) for pTie2, which is consistent with the chemistry step being more rate-limiting for Tie2 as compared to pTie2. The pH-rate profiles of Tie2 and pTie2 revealed a >0.5 unit shift in the pK(a) values of catalytically relevant ionizable residues upon autophosphorylation. The shift in rate-limiting step will result in a different distribution of enzyme pools (e.g., E, E*S, E*P, etc.) which may modulate the susceptibility to inhibition. Tie2 and pTie2 were profiled with a panel of known ATP-competitive kinase inhibitors. Tie2 activation perturbs catalytic residue ionizations, shifts the rate-limiting step to almost exclusive diffusion-control, and transforms the kinase into a more perfect catalyst.

Characterization of a cross-linked DNA-endonuclease VIII repair complex by electrospray ionization mass spectrometry.

Electrospray mass spectrometry techniques were used to characterize components of the active site in Endonuclease VIII by identifying the amino acid sequence and the binding site for a tryptic peptide derived from Endo VIII in a cross-linked DNA-peptide complex. Endo VIII, a DNA repair enzyme with both glycosylase and lyase activities, was covalently bound to a thymidine glycol-containing oligodeoxynucleotide duplex by converting a transient Schiff base formed during the course of the glycosylase activity to a stable covalent bond by chemical reduction with sodium borohydride. After tryptic digestion of the initial product, the identification of the cross-linked peptide was deduced initially from the molecular mass of the tryptic product obtained by negative ion electrospray mass analysis. Nanospray tandem mass spectrometry (MS/MS) analysis of the tryptic product corroborated the molecular mass of the peptide fragment and verified the point of attachment to the oligomer, but failed to produce sufficient fragmentation to sequence the peptide completely. Direct evidence for the amino acid sequence of the peptide was obtained after enzymatic digestion of the DNA portion of the cross-linked DNA-peptide product and analysis by negative ion nanospray MS/MS. Examination of the ions from collision induced fragmentation disclosed that this substance was the N-terminal tryptic fragment of Endo VIII cross-linked to a portion of the oligomer, and that the N-terminal proline from Endo VIII was covalently bound to the residual deoxyribose moiety at the original location of the thymine glycol in the oligomer.

Missense mutations interfere with VEGFR-3 signalling in primary lymphoedema.

Primary lymphoedema is a rare, autosomal dominant disorder that leads to a disabling and disfiguring swelling of the extremities and, when untreated, tends to worsen with time. Here we link primary human lymphoedema to the FLT4 locus, encoding vascular endothelial growth factor receptor-3 (VEGFR-3), in several families. All disease-associated alleles analysed had missense mutations and encoded proteins with an inactive tyrosine kinase, preventing downstream gene activation. Our study establishes that VEGFR-3 is important for normal lymphatic vascular function and that mutations interfering with VEGFR-3 signal transduction are a cause of primary lymphoedema.

Crystal structure of the kinase domain of human vascular endothelial growth factor receptor 2: a key enzyme in angiogenesis.

BACKGROUND: Angiogenesis is involved in tumor growth, macular degeneration, retinopathy and other diseases. Vascular endothelial growth factor (VEGF) stimulates angiogenesis by binding to specific receptors (VEGFRs) on the surface of vascular endothelial cells. VEGFRs are receptor tyrosine kinases that, like the platelet-derived growth factor receptors (PDGFRs), contain a large insert within the kinase domain. RESULTS: We report here the generation, kinetic characterization, and 2.4 A crystal structure of the catalytic kinase domain of VEGF receptor 2 (VEGFR2). This protein construct, which lacks 50 central residues of the 68-residue kinase insert domain (KID), has comparable kinase activity to constructs containing the entire KID. The crystal structure, determined in an unliganded phosphorylated state, reveals an overall fold and catalytic residue positions similar to those observed in other tyrosine-kinase structures. The kinase activation loop, autophosphorylated on Y1059 prior to crystallization, is mostly disordered; however, a portion of it occupies a position inhibitory to substrate binding. The ends of the KID form a beta-like structure, not observed in other known tyrosine kinase structures, that packs near to the kinase C terminus. CONCLUSIONS: The majority of the VEGFR2 KID residues are not necessary for kinase activity. The unique structure observed for the ends of the KID may also occur in other PDGFR family members and may serve to properly orient the KID for signal transduction. This VEGFR2 kinase structure provides a target for design of selective anti-angiogenic therapeutic agents.

Electric fields in active sites: substrate switching from null to strong fields in thiol- and selenol-subtilisins.

Although known to be important factors in promoting catalysis, electric field effects in enzyme active sites are difficult to characterize from an experimental standpoint. Among optical probes of electric fields, Raman spectroscopy has the advantage of being able to distinguish electronic ground-state and excited-state effects. Earlier Raman studies on acyl derivatives of cysteine proteases [Doran, J. D., and Carey, P. R. (1996) Biochemistry 35, 12495-502], where the acyl group has extensive pi-electron conjugation, showed that electric field effects in the active site manifest themselves by polarizing the pi-electrons of the acyl group. Polarization gives rise to large shifts in certain Raman bands, e.g. , the C=C stretching band of the alpha,beta-unsaturated acyl group, and a large red shift in the absorption maximum. It was postulated that a major source of polarization is the alpha-helix dipole that originates from the alpha-helix terminating at the active-site cysteine of the cysteine protease family. In contrast, using the acyl group 5-methylthiophene acryloyl (5-MTA) as an active-site Raman probe, acyl enzymes of thiol- or selenol-subtilisin exhibit no polarization even though the acylating amino acid is at the terminus of an alpha-helix. Quantum mechanical calculations on 5-MTA ethyl thiol and selenol ethyl esters allowed us to identify the conformational states of these molecules along with their corresponding vibrational signatures. The Raman spectra of 5-MTA thiol and selenol subtilisins both showed that the acyl group binds in a single conformation in the active site that is s-trans about the =C-C=O single bond. Moreover, the positions of the C=C stretching bands show that the acyl group is not experiencing polarization. However, the release of steric constraints in the active site by mutagenesis, by creating the N155G form of selenol-subtilisin and the P225A form of thiol-subtilisin, results in the appearance of a second conformer in the active sites that is s-cis about the =C-C=O bond. The Raman signature of this second conformer indicates that it is strongly polarized with a permanent dipole being set up through the acyl group's pi-electron chain. Molecular modeling for 5-MTA in the active sites of selenol-subtilisin and N155G selenol-subtilisin confirms the findings from Raman spectroscopic studies and identifies the active-site features that give rise to polarization. The determinants of polarization appear to be strong electron pull at the acyl carbonyl group by a combination of hydrogen bonds and the field at the N-terminus of the alpha-helix and electron push from a negatively charged group placed at the opposite end of the chromophore.

Structure of Haemophilus influenzae Fe(+3)-binding protein reveals convergent evolution within a superfamily.

The first crystal structure of the iron-transporter ferric ion-binding protein from Haemophilus influenzae (hFBP), at 1.6 A resolution, reveals the structural basis for iron uptake and transport required by several important bacterial pathogens. Paradoxically, although hFBP belongs to a protein superfamily which includes human transferrin, iron binding in hFBP and transferrin appears to have developed independently by convergent evolution. Structural comparison of hFBP with other prokaryotic periplasmic transport proteins and the eukaryotic transferrins suggests that these proteins are related by divergent evolution from an anion-binding common ancestor, not from an iron-binding ancestor. The iron binding site of hFBP incorporates a water and an exogenous phosphate ion as iron ligands and exhibits nearly ideal octahedral metal coordination. FBP is highly conserved, required for virulence, and is a nodal point for free iron uptake in several Gram-negative pathogenic bacteria, thus providing a potential target for broad-spectrum antibacterial drug design against human pathogens such as H. influenzae, Neisseria gonorrhoeae, and Neisseria meningitidis.

Investigation of the impurities found in methamphetamine synthesised from pseudoephedrine by reduction with hydriodic acid and red phosphorus.

The synthesis of methamphetamine from pseudoephedrine via the reduction with hydriodic acid and red phosphorus was studied and the impurities which were generated, along with the methamphetamine, were investigated. Some of the impurities found have been reported previously, while the diastereoisomers of N-methyl-N-(alpha- methylphenethyl)amino-1-phenyl-2-propanone and the cis-cinnamoyl derivative of methamphetamine are reported here for the first time. Further work on the sequence of reactions occurring in this reduction is also reported.

The hair collar sign: marker for cranial dysraphism.

OBJECTIVE: To call attention to a cutaneous marker for neural tube closure defects of the scalp, the "hair collar" sign. This finding consists of a ring of long, dark, coarse hair surrounding a midline scalp nodule. METHODS AND RESULTS: Four children with small congenital scalp nodules and the hair collar sign were studied from the standpoint of clinical findings, radiologic scans, and histology of the excised nodules. All four had an overlying vascular stain in addition to the hair collar. Patients 1 and 2 were found to have encephaloceles, and one had heterotopic brain tissue. The fourth family refused surgery, but the clinical and radiologic findings were consistent with a diagnosis of atretic encephaloceles. One infant had agenesis of the corpus callosum and a Dandy-Walker malformation as associated findings. CONCLUSIONS: The "hair collar" sign should alert the pediatrician to the possibility of ectopic neural tissue in the scalp and/or underlying central nervous system malformations.

Purification and crystallization of a schistosomal glutathione S-transferase.

The 26-kDa glutathione S-transferase from Schistosoma japonica (Sj26), a potential antischistosomal vaccine antigen, has been crystallized in an unligated form. Sj26 was recombinantly produced in E. coli without using a glutathione affinity column to facilitate preparation of unligated enzyme. The recombinant protein contains all 218 residues of Sj26 and an additional 13 residues linked to the C-terminus. Crystals of recombinant Sj26 were obtained by the vapor diffusion method using ammonium sulfate as the precipitant at pH 5.6. The crystals belong to the hexagonal space group P6(3)22 with unit cell dimensions a = b = 125.2 A and c = 72.0 A and contain one Sj26 monomer per asymmetric unit. A complete native diffraction data set has been obtained to 2.4 A resolution.

Crystal structures of a schistosomal drug and vaccine target: glutathione S-transferase from Schistosoma japonica and its complex with the leading antischistosomal drug praziquantel.

Glutathione S-transferase (GST), an essential detoxification enzyme in parasitic helminths, is a major vaccine target and an attractive drug target against schistosomiasis and other helminthic diseases. Crystal structures of the 26 kDa GST from the helminth Schistosoma japonica (SjGST) have been determined for the unligated enzyme (resolution = 2.4 A, R-factor = 19.7%) and for the enzyme bound to the leading antischistosomal drug praziquantel (resolution = 2.6 A, R-factor = 21.2%). The protein, recombinantly expressed using the Pharamacia PGEX-3X vector for production of GST fusion proteins, contains all 218 residues of SjGST and an additional 13 residues at the C terminus. The structure of unligated SjGST shows that the glutathione binding site pre-exists unchanged in the ligand-free enzyme and is conserved between parasitic and the mammalian class mu enzymes. At therapeutic concentrations the leading antischistosomal drug praziquantel (PZQ) binds one drug per enzyme homodimer in the dimer interface groove adjoining the two catalytic sites. This establishes a protein target for PZQ, identifies the GST non-substrate ligand transport site, and implicates PZQ in steric inhibition of SjGST catalytic and transport for large ligands. Thus, increased expression or mutagenesis of SjGST by the parasite may confer resistance to PZQ. Differences in the xenobiotic binding region between parasitic and mammalian GSTs reveal a distinct substrate repertoire for SjGST and, together with the newly identified PZQ binding site, provide the basis for design of novel antischistosomal drugs. Due to the widespread use expression systems based on SjGST fusions, the atomic structure of SjGST should also provide an important tool for phasing fusion protein structures by molecular replacement.

Pharmacokinetics of pretargeted monoclonal antibody 2D12.5 and 88Y-Janus-2-(p-nitrobenzyl)-1,4,7,10-tetraazacyclododecanetetraacetic acid (DOTA) in BALB/c mice with KHJJ mouse adenocarcinoma: a model for 90Y radioimmunotherapy.

Three-step pretargeting for radioimmunotherapy in BALB/c mice with KHJJ tumors was done with monoclonal antibody (mAb) 2D12.5, which is specific for yttrium-1,4,7,10-tetraazacyclododecanetetraacetic acid (DOTA) but nonspecific for the tumor. Tumor uptake was by passive diffusion of mAb through leaky neovasculature in the tumor. The three steps were: (a) anti-hapten mAb 2D12.5 (0 h); (b) polyvalent haptenprotein conjugate chase (20 h); and (c) 88Y-labeled monovalent DOTA or bivalent Janus-DOTA haptens (21 h) and organ and tumor bioassay (24 h). Rapid tumor (T) uptake and high tumor:blood ratio (T:BL) was seen 3 h after injection after step c. For monovalent 88Y-DOTA, T = 1.7%/g* and T:BL = 16:1; for bivalent 88Y-Janus-DOTA, T = 4.41%/g* and T:BL = 21:1 at 3 h (*, P < 0.001). Blood and bone plus marrow were << 1%/g, and liver was < 1%/g. The 24-h whole body retention was approximately 5% of injected dose with 1% in tumor (20% of total), 1.8% in other organs, and 2.2% in carcass; the 24-h whole body retention of covalent nonspecific antibody conjugates was > 80% of injected dose. The biological half-life in the tumor of 0.9 microCi 88Y-Janus-DOTA was approximately 24 h, measured daily for 5 days. Activity in microCi/g of tumor and blood for 90Y equimolar to the amount of 88Y injected (0.9 microCi 88Y = 0.744 pmol = 36.47 microCi 90Y) was used for calculating the area under the curve of tumor and blood in microCi-h/g of 90Y. The 90Y radiation absorbed dose (RAD) from multiplying microCi-h/g x the 90Y absorbed dose constant, 1.99 RAD-g/microCi-h, gave T = 89 RAD and BL = 3.7 RAD. The therapeutic ratio from RAD T:RAD BL = 24:1. These results indicate that pretargeting 90Y hapten-specific mAb for radioimmunotherapy has considerable promise.

Immunogenicity in rabbits and mice of an antibody-chelate conjugate: comparison of (S) and (R) macrocyclic enantiomers and an acyclic chelating agent.

The macrocyclic bifunctional chelating agent 2-(p-bromoacetamidobenzyl)-1,4,7,10-tetraazacyclododecanetetraa cetic acid (BAD), forms inert metal complexes ideal for radioimmunotherapy. Kosmas et al. (Cancer Res., 52: 904-911, 1992) found 2-imminothiolane linker-(S)-BAD monoclonal antibody HMFG1 highly immunogenic in patients. We studied the immunogenicity of (S) and (R) enantiomers of 2-imminothiolane linker-BAD rabbit IgG, monoclonal antibody Lym-1, and Lym-1 2-imminothiolane linker-(S)-bromoacetamidobenzyl-EDTA in 15 rabbits. Five groups of three each were given 0.1, 1.0, or 10 mg of 111In conjugate i.v., blood samples were taken daily for 14 days and biweekly for 70 days, and the plasma T1/2 was calculated. A drop in plasma 111In at 6-8 days coincided with the appearance of antibody on enzyme-linked immunosorbent assay. Specific anti-(S)-BAD, anti-(R)-BAD, anti-(S)-bromoacetamidobenzyl-EDTA, and anti-mouse IgG were measured. Rabbit IgG conjugates did not elicit an immune response. Mouse IgG conjugates were immunogenic on the first exposure, with both anti-1,4,7,10-tetraazacylododecane N,N',N'',N'''-tetraacetic acid and anti-mouse responses. Anti-1,4,7,10-tetraazacylododecane N,N',N'',N'''-tetraacetic acid was specific for the (S) or (R) enantiomer, but cross-reaction appeared with reboosting. A second injection of the opposite enantiomer gave a response to that enantiomer. Lym-1 bromoacetamidobenzyl-EDTA produced anti-bromoacetamidobenzyl-EDTA and anti-mouse response.

Viability and biodistribution of 68Ga MPO-labelled human platelets.

The viability and biodistribution of 68Ga-mercaptopyridine-N-oxide (MPO)-labelled autologous platelets was studied in 10 patients. The average platelet labelling yield was 36 +/- 12% and injected activity was 2.0 +/- 0.9 mCi 68Ga. The % activity in platelets per ml whole blood was 64 +/- 20% at 15 min-1.0 h postinjection and 76 +/- 14% at 2-4h. The average recovery of platelets (% injected platelets circulating in peripheral blood) was 31 +/- 21% at 15 min-1 h and 39 +/- 20% at 2-4 h. The positron emission tomographic (PET) images showed high circulating vascular background. Two patients had technically inadequate scans, and six were false negative due to high blood background. One patient with a massive pulmonary embolus occurring 24 h prior to scanning had marked uptake of 68Ga platelets in a large clot in the superior branch of the right main pulmonary artery. A second patient, with 68Ga platelets circulating during angioplasty of a left posterior tibial artery stenosis, had intense uptake in the lesion shown on the PET scan obtained 4 h following the procedure. These results indicate good viability of 68Ga-MPO-labelled autologous human platelets, but poor visualization of clots by PET imaging, due to the high blood background at early times.

Crystal structures of chicken liver dihydrofolate reductase: binary thioNADP+ and ternary thioNADP+.biopterin complexes.

The role of the 3'-carboxamide substituent of NADPH in the reduction of pteridine substrates as catalyzed by dihydrofolate reductase (EC 1.5.1.3, DHFR) has been investigated by determining crystal structures at 2.3 A of chicken liver DHFR in a binary complex with oxidized thionicotinamide adenine dinucleotide (thioNADP+) and in a ternary complex with thioNADP+ and biopterin. These structures are isomorphous with those previously reported for chicken liver DHFR [Volz, K.W., Matthews, D.A., Alden, R.A., Freer, S. T., Hansch, C., Kaufman, B. T., & Kraut, J. (1982) J. Biol. Chem. 257, 2528-2536]. ThioNADPH, which has a 3'-carbothioamide substituent in place of a 3'-carboxamide, functions very poorly as a coenzyme for DHFR [Williams, T. J., Lee, T. K., & Dunlap, R. B. (1977) Arch, Biochem. Biophys. 181, 569-579; Stone, S. R., Mark, A., & Morrison, J. F. (1984) Biochemistry 23, 4340-4346]. Comparisons show that, while NADP+ and NADPH bind to DHFR with the pyridine ring and 3'-carboxamide coplanar, the thioamide group is twisted by 23 degrees from the pyridine plane in both the binary and ternary complexes. This twist appears to be due to steric conflict between the thioamide sulfur atom and both the pyridine ring at C4 and the adjacent protein backbone at Ala-9. It results in an unfavorably close contact between the sulfur and the biopterin pteridine ring (0.9 A less than the van der Waals separation) which, on the basis of the refined structure, greatly destabilizes the binding of biopterin.(ABSTRACT TRUNCATED AT 250 WORDS)

Pretargeted immunoscintigraphy: effect of hapten valency on murine tumor uptake.

A method of radioimmunoscintigraphy using bivalent "Janus" haptens with an apparent enhanced affinity ("avidity") for the antibody is described. Janus with 50 micrograms pretargeted Mab WC3A11 resulted in significantly higher murine tumor concentrations (approximately 7%/g) compared to monovalent haptens (approximately 1.4%/g, p < 0.001), and the same high tumor-to-background ratios (approximately 3/1). Janus was synthesized by coupling two molecules of BABE together with a 1,4 butanedithiol linker. Janus itself was rapidly excreted (T1/2b = 42 min) by the kidneys and did not concentrate in any other organs or tissues. Three-step pretargeted immunoscintigraphy (binder, chaser, tracer) with 111In- or 67Ga-Co(III) Janus produced excellent mouse tumor images in 3 hr with high tumor-to-background ratios. The use of short-lived tracers, such as 99mTc and 68Ga, with a T1/2p of hours to image antibodies that localize slowly over several days in vivo is accessible with this new technology.

Crystal structure of chicken liver dihydrofolate reductase complexed with NADP+ and biopterin.

The 2.2-A crystal structure of chicken liver dihydrofolate reductase (EC 1.5.1.3, DHFR) has been solved as a ternary complex with NADP+ and biopterin (a poor substrate). The space group and unit cell are isomorphous with the previously reported structure of chicken liver DHFR complexed with NADPH and phenyltriazine [Volz, K. W., Matthews, D. A., Alden, R. A., Freer, S. T., Hansch, C., Kaufman, B. T., & Kraut, J. (1982) J. Biol. Chem. 257, 2528-2536]. The structure contains an ordered water molecule hydrogen-bonded to both hydroxyls of the biopterin dihydroxypropyl group as well as to O4 and N5 of the biopterin pteridine ring. This water molecule, not observed in previously determined DHFR structures, is positioned to complete a proposed route for proton transfer from the side-chain carboxylate of E30 to N5 of the pteridine ring. Protonation of N5 is believed to occur during the reduction of dihydropteridine substrates. The positions of the NADP+ nicotinamide and biopterin pteridine rings are quite similar to the nicotinamide and pteridine ring positions in the Escherichia coli DHFR.NADP+.folate complex [Bystroff, C., Oatley, S. J., & Kraut, J. (1990) Biochemistry 29, 3263-3277], suggesting that the reduction of biopterin and the reduction of folate occur via similar mechanisms, that the binding geometry of the nicotinamide and pteridine rings is conserved between DHFR species, and that the p-aminobenzoylglutamate moiety of folate is not required for correct positioning of the pteridine ring in ground-state ternary complexes. Instead, binding of the p-aminobenzoylglutamate moiety of folate may induce the side chain of residue 31 (tyrosine or phenylalanine) in vertebrate DHFRs to adopt a conformation in which the opening to the pteridine binding site is too narrow to allow the substrate to diffuse away rapidly. A reverse conformational change of residue 31 is proposed to be required for tetrahydrofolate release.