Search PubMed⌕ Search

Biomedical subjects

C W Heizmann

Publications and source records attributed to C W Heizmann.

At least 109 records · Page 6Linked to original sources
PubMed1995

Amino acid sequence analysis of human S100A7 (psoriasin) by tandem mass spectrometry.

The Ca(2+)-binding proteins regulate a number of cellular and extracellular activities and deregulations of S100 gene expression are associated with several human diseases. For example, S100A7 is upregulated in psoriatic skin, implicating a link with psoriasis, a chronic inflammatory dermatosis. We purified human S100A7 and determined its protein sequence by tandem mass spectrometry and Edman microsequence analysis. Interestingly, a sequence comparison of S100A7 with all known human S100 proteins showed that S100A7 is the most divergent of all S100 proteins.

Amino Acid Sequence↗
PubMed1995

6-Pyruvoyl tetrahydropterin synthase, an enzyme with a novel type of active site involving both zinc binding and an intersubunit catalytic triad motif; site-directed mutagenesis of the proposed active center, characterization of the metal binding site and modelling of substrate binding.

6-Pyruvoyl tetrahydropterin synthase (PTPS) is an enzyme involved in tetrahydrobiopterin biosynthesis, the cofactor for several aromatic amino acid monooxygenases and the nitric oxide synthases. The crystal structure of PTPS was recently solved and showed a homohexameric enzyme composed of a dimer of trimers. A transition metal binding site formed by the three histidine residues 23, 48 and 50 was found in each subunit. We showed by metal analysis and reconstitution of apo-PTPS that Zn(II) was the bound transition metal and responsible for the enzymatic activity. Site-directed mutagenesis of each of these three histidine residues resulted in a complete loss of metal binding and enzymatic activity. The three residues, Cys42, His89 and Glu133, located close to the metal binding site, were previously postulated to be involved in the catalytic reaction. We altered these residues and found a complete loss of enzymatic activity for the mutant C42A. The two mutants, H89N and E133Q, showed 4.3% and 1.3% enzymatic activity, respectively, but had similar KM values for the substrate as compared to wild-type PTPS. Based on these results we propose a model of the substrate fitted into the active site and we described a novel intersubunit catalytic triad motif composed of the amino acid residues Cys42, His89 and Asp88. Different from most other catalytic triads that catalyse the hydrolysis of an amide or ester bond, the catalytic triad in the active site of PTPS seems to be involved in the deprotonation of the substrate's side-chain carbons. Our model also proposes Zn(II) as the coordination site for the two substrate side-chain hydroxy groups as well as the involvement of Glu133 as putative stereospecific proton server.

Alcohol Oxidoreductases↗
PubMed1995

Purification and cation binding properties of the recombinant human S100 calcium-binding protein A3, an EF-hand motif protein with high affinity for zinc.

The calcium-binding protein S100A3 is an unusual member of the S100 family, characterized by its very high content of Cys. In order to study the biochemical, cation-binding, and conformational properties, we produced and purified the recombinant human protein in Escherichia coli. The recombinant protein forms noncovalent homodimers, tetramers, and polymers in vitro with a subunit molecular weight of 11,712. The Zn(2+)-binding parameters of S100A3 were studied by equilibrium gel filtration and yielded a stoichiometry of four Zn2+ per monomer with a [Zn2+]0.5 of 11 microM and a Hill coefficient of 1.4 at physiological ionic strength. The affinity for Ca2+ is too low to be determined by direct methods (KCa > 10 mM). Ca(2+)- and Zn(2+)-binding can be followed by optical methods: the Trp-45 fluorescence is high in the metal-free form and addition of Zn2+ and Ca2+, but not of Mg2+, leads to a 4-fold quenching. Ca2+ and Zn2+ promote also quite similar conformational changes in the Tyr and Trp environment as monitored by difference spectrophotometry. Fluorescence titrations with Zn2+ confirmed that there is one set of high affinity binding sites with a [Zn2+]0.5 of 8 microM and a Hill coefficient of 1.3. Binding of Zn2+ to a second set of low affinity sites induces protein precipitation. Fluorescence titrations with Ca2+ confirmed the very low affinity of S100A3 for this ion with a [Ca2+]0.5 of 30 mM and slight negative cooperativity. Mg2+ has no effect on this binding curve. Of the 10 Cys residues in S100A3, 5 only are free thiols, and accessible to 5,5'-dithiobis(2-nitro-benzoic acid); they display a high reactivity in the metal-free and Ca2+ form, but a 20-fold lowered reactivity in the Zn2+ form of S100A3. Ca(2+)-binding promotes the formation of a solvent-accessible hydrophobic surface as monitored by the 60-fold fluorescence increase of 2-p-toluidinylnaphthalene-6-sulfonate, whereas Zn2+ has no noticeable influence. Our data indicate that Ca2+ and Zn2+ do not bind to the same sites and that under physiological conditions S100A3 is a Zn(2+)-binding rather than a Ca(2+)-binding protein; nevertheless, very specific conformational changes are introduced by either Ca2+ or Zn2+. Since no Zn(2+)-binding motif of known structure was identified in the primary sequence of S100A3, the results are suggestive for a novel Zn(2+)-binding motif.

Base Sequence↗
PubMed1995

Human pterin-4 alpha-carbinolamine dehydratase/dimerization cofactor of hepatocyte nuclear factor-1 alpha. Characterization and kinetic analysis of wild-type and mutant enzymes.

Pterin-4a-carbinolamine dehydratase/dimerization cofactor for hepatocyte nuclear factor-1 alpha is a protein with two different functions. We have overexpressed and purified the human wild-type protein, and its Cys81Ser and Cys81Arg mutants. The Cys81Arg mutant has been proposed to be causative in a hyperphenylalaninaemic patient [Citron, B. A., Kaufman, S., Milstien, S., Naylor, E. W., Greene, C. L. & Davis, M. D. (1993) Am. J. Hum. Genet. 53, 768-774]. The dehydratase behaves as a tetramer on gel filtration, while cross-linking experiments showed mono-, di-, tri-, and tetrameric forms, irrespective of the presence of the single Cys81. Sulfhydryl-modifying reagents did not affect the activity, but rather showed that Cys81 is exposed. Various pterins bind and quench the tryptophan fluorescence suggesting the presence of a specific binding site. The fluorescence is destroyed upon light irradiation. Wild-type and the Cys81Ser protein enhance the rate of the phenylalanine hydroxylase assay approximately 10-fold, a value similar to that of native dehydratase from rat liver; the Cys81Arg mutant, in contrast, has significantly lower activity. This is compatible with the hypothesis that the dehydratase is a rate-limiting factor for the in vivo phenylalanine hydroxylase reaction. The three proteins enhance the spontaneous dehydration of the synthetic substrate 6,6-dimethyl-7,8-dihydropterin-4a-carbinolamine approximately 50-70-fold at 4 degrees C and pH 8.5. The results are discussed in view of the recently solved three-dimensional structure of the enzyme [Ficner, R., Sauer, U. W., Stier, G. & Suck, D. (1995) EMBO J. 14, 2032-2042].

Base Sequence↗
PubMed1995

Characterization of the human PCBD gene encoding the bifunctional protein pterin-4 alpha-carbinolamine dehydratase/dimerization cofactor for the transcription factor HNF-1 alpha.

The human pterin-4 alpha-carbinolamine dehydratase (PCD)/dimerization cofactor for the transcription factor HNF-1 alpha is a bifunctional protein proposed to be involved in entirely different biochemical functions. We previously established the complete amino acid sequence for the human liver PCD and subsequently isolated its corresponding cDNA. Using this cDNA as a probe, we isolated and determined the complete nucleotide sequence and flanking regions of the single human PCBD gene. The protein coding region of the gene is about 5 kb in length and contains 4 exons. We also defined the messenger RNA 5'-end by reverse transcription of the cap structure, thus allowing to analyze the promoter organization. Within the 5'-flanking sequence, potential regulatory regions include consensus binding sites for transcription factor Sp1, an AP-1, and several AP-2 binding sites; however, the 5' upstream region lacks both a proximal TATA and CAAT box promoter element.

Amino Acid Sequence↗
PubMed1995

Isolation of a YAC clone covering a cluster of nine S100 genes on human chromosome 1q21: rationale for a new nomenclature of the S100 calcium-binding protein family.

S100 proteins are low-molecular-weight calcium-binding proteins of the EF-hand superfamily and appear to be involved in the regulation of a number of cellular processes such as cell cycle progression and differentiation. More than 10 members of the S100 protein family have been described from human sources so far. We have now isolated a YAC clone from human chromosome 1q21, on which 9 different genes coding for S100 calcium-binding proteins could be localized. Moreover, we have mapped the gene coding for S100P to human chromosome 4p16 and thereby completed the chromosomal assignments of all known human S100 genes. The clustered organization of S100 genes in the 1q21 region allows us to introduce a new logical nomenclature for these genes, which is based on the physical arrangement on the chromosome. The new nomenclature should facilitate and further the understanding of this protein family and be easily expandable to other species.

Base Sequence↗
PubMed1995

A novel EF-hand Ca(2+)-binding protein from abdominal muscle of crustaceans with similarity to calcyphosine from dog thyroidea.

The amino acid sequence of a novel EF-hand Ca(2+)-binding protein from the abdominal muscle of the crayfish, Orconectes limosus, has been elucidated by tandem mass spectrometry and automated Edman degradation. The name CCBP-23 (23-kDa crustacean Ca(2+)-binding protein) is proposed. The protein can also exist as a disulfide-linked homodimer. The sequence of the monomeric form spans 200 residues with an acetylated N-terminal Ser and reveals four EF-hand domains. The 174-mass-unit difference between the calculated average molecular mass of 22,669.6 Da deduced from the sequence and the obtained electrospray ionization mass spectroscopy (ESI-MS) mass of 22,844 Da has not yet been explained. Partial sequence analysis (137 residues) of CCBP-23 from the lobster, Homarus americanus, showed a sequence identity of 74% with the crayfish protein. Homology searches revealed a 44% sequence identity of CCBP-23 from crayfish to calcyphosine, a Ca(2+)-binding protein from dog thyroidea (Lefort et al., 1989). Although CCBP-23 also shows a 44% identity to R2D5 antigen (Nemoto et al., 1993), we believe that both proteins represent two distinct subgroups within the family of EF-hand proteins.

Amino Acid Sequence↗
PubMed1995

Distribution of the calcium binding proteins, calbindin D-28K and parvalbumin, in the subicular complex of the adult mouse.

The immunohistochemical localizations of two specific calcium binding proteins, calbindin D-28K (calbindin) and parvalbumin (PV) were examined in the subicular complex, that is, the subiculum, presubiculum, and parasubiculum, of the adult mouse and were compared in detail with staining pattern of the acetylcholinesterase (AChE) histochemistry. The calbindin immunoreactivity exhibited a conspicuous regional and laminar pattern of distribution, which somewhat resembled the AChE staining pattern but was apparently different from the latter in various points. The PV immunoreactivity also exhibited a characteristic regional difference, although less prominent. The subiculum could be divided into two subregions, intensely calbindin-immunoreactive (calbindin-IR) and AChE stained proximal subiculum and only faintly calbindin-IR and AChE stained distal subiculum. In the subiculum most of calbindin-IR neurons were pyramidal cells which were clustered in the superficial half of the cell layer in the proximal subiculum and appeared to be segregated from calbindin negative pyramidal cells located in the distal subiculum and in the basal part of the proximal subiculum. In the presubiculum calbindin-IR neurons were clustered in layer 2, most of which were supposed to be presubicular pyramidal cells. In the parasubiculum, the overall immunostaining pattern of PV and calbindin were somewhat complementary. In the transition area calbindin-IR neurons were clustered but few PV-IR neurons were located, and thus the distribution of immunoreactive neuronal somata was apparently different from the adjacent parts of the parasubiculum, indicating that the transition area might be a separate entity. In addition to calbindin-IR presumable principal neurons, calbindin-IR and PV-IR nonpyramidal cells were scattered throughout the subicular complex. Furthermore, these two calcium binding proteins were colocalized in some nonpyramidal cells in the subicular complex. The present study revealed some new aspects of the areal and laminar organization of the subicular complex, which had not been shown by previous classical purely morphological approaches.

Acetylcholinesterase↗
PubMed1995

A missense mutation in a patient with guanosine triphosphate cyclohydrolase I deficiency missed in the newborn screening program.

A patient with guanosine triphosphate cyclohydrolase I deficiency passed the newborn phenylketonuria screening program. The characteristic clinical phenotype developed in a 5-month-old patient; elevated plasma phenylalanine, undetectable urinary pterins, and absence of the enzyme activity in a liver biopsy were present. A point mutation that results in an amino acid substitution from methionine to isoleucine at position 211 was proposed to be the cause for this new phenotypic expression of guanosine triphosphate cyclohydrolase I deficiency.

Base Sequence↗
PubMed1995

Localization of the Ca(2+)-binding alpha-parvalbumin and its mRNA in epiphyseal plate cartilage and bone of growing rats.

This study describes the localization of alpha-parvalbumin, in undecalcified tibial epiphyseal cartilage and bone of growing rats by immunocytochemistry in the light microscope, and of parvalbumin mRNA by in situ hybridization. They were compared to the distribution of the calbindin-D9K and its mRNA in rat epiphyseal cartilage. All the chondrocytes of the epiphyseal cartilage were parvalbumin-immunopositive, but there was no parvalbumin immunoreactivity in the uncalcified or calcified extracellular cartilage matrix. The intensity of the immunostaining increased from the resting and proliferative to the mature and hypertrophic chondrocytes, with the greatest intensity in the terminal hypertrophic chondrocytes in the calcifying zone. The parvalbumin immunostaining was located in the cytoplasm, but no immunoreactivity was detected in any chondrocyte processes. The parvalbumin mRNA distribution and levels, as revealed by in situ hybridization, exactly mirrored those of the parvalbumin protein. In contrast to parvalbumin, calbindin-D9K and its mRNA appeared in mature chondrocytes and decreased in hypertrophic up to calcifying chondrocytes. Calbindin-D9K was located in the cytoplasm and all along the cell processes. In bone, the osteoblasts and the osteocytes of trabecular and compact cortical bones were immunoreactive for parvalbumin and contained parvalbumin mRNA. Parvalbumin lay in their cytoplasm, but there was no parvalbumin immunostaining in the extracellular uncalcified or mineralized bone matrix. The long processes of osteocytes, in compact bone only, were parvalbumin immunoreactive. Osteoclasts contained cytoplasmic parvalbumin immunoreactivity. Thus, the pattern of immunoreactive parvalbumin distribution indicates that the protein is not involved in the extracellular mineralization of cartilage and bone matrix. It appears to be associated with specific calcium-related intracellular functions in chondrocytes and in osteoblasts, osteocytes, and osteoclasts. As the highest cytoplasmic concentration of parvalbumin is in the terminal hypertrophic chondrocytes, parvalbumin could act as a calcium buffer to delay the death of chondrocytes. In compact bone, parvalbumin could also have a role throughout the osteocyte processes in regulating the fluxes of calcium ions for mineral homeostatis.

Animals↗
PubMed1995

Chemically defined neuron groups and their subpopulations in the glomerular layer of the rat main olfactory bulb.

Chemically-defined neuron groups and their subpopulations in the glomerular layer of the rat main olfactory bulb were revealed immunocytochemically using antibodies against gamma-amino butyric acid (GABA), tyrosine hydroxylase (TH), methionin-enkephalin-Arg6-Gly7-Leu8 (ENK), calretinin (CR), calbindin-D28K (calbindin) and thyrotropin-releasing hormone (TRH). GABA-like immunoreactive (GABA-LIR) neurons and CR immunoreactive (CR-IR) neurons were most numerous; they were about 1.5-3 times more numerous than calbindin immunoreactive (calbindin-IR), TH immunoreactive (TH-IR), ENK-like immunoreactive (ENK-LIR) and THR-like immunoreactive (TRH-LIR) neurons. We identified at least three distinct chemically-defined neuron groups, GABA-LIR neurons, CR containing neurons and calbindin containing neurons, since these three neuron groups were almost separate from one another. On the other hand, TH-IR and ENK-LIR neurons were nearly included in and thus considered to be subpopulations of GABA-LIR and CR-IR neurons, respectively, for about 80% of these two neuron groups contained GABA-L and CR immunoreactivities, respectively. TRH-LIR neurons appeared to be divided into two subpopulations, one containing the GABA-L immunoreactivity and the other containing the CR immunoreactivity. Thus in the glomerular layer of the rat olfactory bulb, GABA-LIR, CR-IR and calbindin-IR cells could be considered to be three distinct chemically-defined neuron groups, whereas TH-IR, TRH-LIR and ENK-LIR neurons were regarded as their subpopulations. Furthermore, some neurons groups, whereas TH-IR, TRH-LIR and ENK-LIR neurons were regarded as their subpopulations. Furthermore, some neurons are supposed to contain three substances (e.g. GABA + TH + TRH, GABA + TRH + EnK, CR + TRH + ENK, GABA + TRH + CR) or a few might even contain four substances (e.g. GABA + TRH + CR + ENK). Preliminary quantitative analysis using the optical disector method showed percentages of these three main neuron groups to total cells in the glomerular layer; that is, neuron groups containing GABA, CR and calbindin were about 20%, 20% and 10%, respectively.

Animals↗
PubMed1994

Expression of intracellular calcium-binding proteins in cultured skin fibroblasts from Alzheimer and normal aged donors.

Disturbed calcium homeostasis may play a role in the etiology in Alzheimer's and other neurodegenerative diseases. A protective role against cellular degeneration has been postulated for Ca(2+)-binding proteins in certain neuron populations. Recent data suggest that intracellular free calcium regulation is also altered in several non-neuronal cells, including skin fibroblasts, from patients with Alzheimer's disease. In this study we analyzed the expression of several EF-hand Ca(2+)-binding proteins in cultured skin fibroblasts from Alzheimer patients and age-matched normal donors. We detected a strong expression of some members of the S100 Ca(2+)-binding protein family and of calcineurin A. However, no significant differences were found between both types of donors by Northern blot and Western blot analysis. In addition, similar signals were detected on 45Ca(2+)-blots of fibroblasts extracts of Alzheimer patients and control donors. The present findings indicate that the altered level of some intracellular calcium-binding proteins in certain brain areas of Alzheimer patients is not found in skin fibroblasts of these patients.

Aged↗
PubMed1994

Synaptic contacts between mitral/tufted cells and GABAergic neurons containing calcium-binding protein parvalbumin in the rat olfactory bulb, with special reference to reciprocal synapses between them.

It has been believed so far that in the mammalian olfactory bulb, only granule cells form reciprocal synapses with mitral/tufted cells in the external plexiform layer (EPL). However, the present electron microscopic study demonstrated for the first time that another group of immunocytochemically defined interneurons different from granule cells, viz., neurons containing a specific calcium-binding protein parvalbumin, also made reciprocal synapses with mitral/tufted cells in the EPL.

Animals↗
PubMed1994

Expression of Ca(2+)-binding proteins of the S100 family in malignant human breast-cancer cell lines and biopsy samples.

In order to examine whether the expression of calcium-binding proteins of the S100 family may correlate with the transformation grade of human mammary-tumor cells, we studied the expression patterns of 4 members of this family (CACY, CAPL, S100L, S100 alpha/beta) in human breast-cancer cell lines. Each S100 protein is shown to be individually regulated in the human breast-cancer cell lines we studied, but it appears that the expression levels of S100 proteins do not strictly correlate with prognostic factors or the tumorigenicity of the cells. However, 2 aggressive cell lines, MDA-MB-231 and HS-578T, show elevated expression of CAPL. We show that methylation may account for partial regulation of the S100 genes, whereas neither genomic rearrangements in the S100 gene cluster region nor gene dosis effects seem to influence their expression pattern in MDA-MB-231 and MCF-7 cells. On the basis of our genomic analyses, we can localize the gene for S100L within 5 kb upstream of S100E, thus extending the S100 gene cluster by one gene. A series of primary breast tumors was collected and tested for expression of CAPL, CACY and S100 alpha/beta. The results show that all human breast-cancer tissues tested express CACY, whereas the presence of CAPL is more restricted. There is a significant correlation between enhanced expression of CAPL and presence of the invasivity marker urokinase-type plasminogen activator (uPA). This observation suggests that CAPL may play an important role in the acquisition of metastatic potential of human mammary epithelial cells.

Breast Neoplasms↗
PubMed1994

Purification and characterization of the recombinant human calcium-binding S100 proteins CAPL and CACY.

The S100 proteins CAPL and CACY are expressed in a tissue- and cell-specific manner and have been reported to be associated with the metastatic phenotype of tumor cells. In order to study the biochemical, cation-binding, and conformational properties, we produced and purified large amounts of the recombinant human proteins in Escherichia coli. Several characteristics of native proteins are shown to correspond to those of the bacterially expressed proteins. Both are able to form homodimers in vitro, probably the biologically active species, but not heterodimers. The Ca(2+)-binding parameters were studied by flow offlysis at physiological ionic strength. Both isotherms show a maximum of two Ca2+ per protein and are insensitive to Mg2+, indicating that the sites are of the Ca(2+)-specific type. The isotherms show slight (CAPL, nH = 1.15) or pronounced (CACY, nH = 1.33) positive cooperativity with K0.5 values of 0.32 mM (CACY) and 0.15 mM (CAPL), indicating that the sites are of the low-affinity type. Conformational changes in the Tyr microenvironment of CACY indicate that Ca2+ binding induces a shift of Tyr to a less polar environment. Mg2+ does not affect the fluorescence properties nor does it induce a difference spectrum, thus suggesting that at physiological ionic conditions it does not interact with the protein. The Ca(2+)-induced difference spectra of CAPL are about 3 times smaller than those of CACY, suggesting that the additional Tyr84 in CACY is much more sensitive to Ca2+ than the two Tyr residues conserved in both proteins.

Animals↗
PubMed1994

Characterization and primary structure of amphioxus troponin C.

Troponin C (TnC) from amphioxus (Protochordate) was purified and its primary structure determined. Unlike the case of vertebrates and other invertebrates, amphioxus TnC is found in the soluble fraction after extractions at physiological ionic strength in the presence of Ca2+. Edman sequencing combined with mass spectroscopy indicate that the protein contains 163 amino acid residues. It possesses an acetylated N-terminus (although a small percentage has a free Ser N-terminus) and either epsilon-N-methyllysine or epsilon-N-dimethyllysine in position 20. It displays about 50% sequence identity with vertebrate skeletal-muscle and cardiac-muscle TnC, 44% with TnC of sea squirt, also a Protochordate, and 30% with other invertebrate TnC. Like vertebrate TnC, amphioxus TnC contains a N-terminal alpha-helix plus the usual four ancestral Ca(2+)-binding regions, but analysis of the sequence suggests that the fourth site is not functional. Flow dialysis shows that amphioxus TnC binds three Ca2+ with the mean apparent affinity constant K' of 3.4 +/- 1.5 10(5) M-1. No cooperativity exists between the sites, and the presence of up to 10 mM Mg2+ does not influence the Ca(2+)-binding isotherm, indicating that the metal-binding sites are Ca(2+)-specific at physiological Mg2+ concentrations. It forms a Ca(2+)-dependent, 1:1 complex with melittin and rabbit or crayfish troponin I (TnI). Amphioxus TnC possesses one Trp residue in position 151 and one at the C-terminus. Trp fluorescence suggests that one or both residues are solvent-exposed in the metal-free form and efficiently shielded in the Ca2+ form. Although Mg2+ has no effect on the Ca2+ binding, the Trp fluorescence is influenced by millimolar Mg2+, suggesting the presence of one or more independent Mg(2+)-binding site(s). A phylogenetic analysis clearly shows that amphioxus TnC is positioned on the branch of the Chordates, but at a distance from the vertebrate TnC. Its place on the phylogenetic tree is in accordance with the consensus evolutionary phylogeny.

Amino Acid Sequence↗
PubMed1994

Three-dimensional structure of 6-pyruvoyl tetrahydropterin synthase, an enzyme involved in tetrahydrobiopterin biosynthesis.

The crystal structure of rat liver 6-pyruvoyl tetrahydropterin synthase has been solved by multiple isomorphous replacement and refined to a crystallographic R-factor of 20.4% at 2.3 A resolution. 6-Pyruvoyl tetrahydrobiopterin synthase catalyses the conversion of dihydroneopterin triphosphate to 6-pyruvoyl tetrahydropterin, the second of three enzymatic steps in the synthesis of tetrahydrobiopterin from GTP. The functional enzyme is a hexamer of identical subunits. The 6-pyruvoyl tetrahydropterin synthase monomer folds into a sequential, four-stranded, antiparallel beta-sheet with a 25 residue, helix-containing insertion between strands 1 and 2 at the bottom of the molecule, and a segment between strands 2 and 3 forming a pair of antiparallel helices, layered on one side of the beta-sheet. Three 6-pyruvoyl tetrahydropterin synthase monomers form an unusual 12-stranded antiparallel beta-barrel by tight association between the N- and C-terminal beta-strands of two adjacent subunits. The barrel encloses a highly basic pore of 6-12 A diameter. Two trimers associate in a head-to-head fashion to form the active enzyme complex. The substrate-binding site is located close to the trimer-trimer interface and comprises residues from three monomers: A, A' and B. A metal-binding site in the substrate-binding pocket is formed by the three histidine residues 23, 48 and 50 from one 6-pyruvoyl tetrahydropterin synthase subunit. Close to the metal, but apparently not liganding it, are residues Cys42, Glu133 (both from A) and His89 (from B), which might serve as proton donors and acceptors during catalysis.

Alcohol Oxidoreductases↗