Clinical, biochemical and genetic findings in adult patients with chronic hypophosphatasemia.

PURPOSE: The study aimed to define the clinical, biochemical and genetic features of adult patients with osteopenia/osteoporosis and/or bone fragility and low serum alkaline phosphatase (sALP). METHODS: Twenty-two patients with at least two sALP values below the reference range were retrospectively enrolled after exclusion of secondary causes. Data about clinical features, mineral and bone markers, serum pyridoxal-5'-phosphate (PLP), urine phosphoethanolamine (PEA), lumbar and femur bone densitometry, and column X-ray were collected. Peripheral blood DNA of each participant was analyzed to detect ALPL gene anomalies. RESULTS: Pathogenic ALPL variants (pALPL) occurred in 23% and benign variants in 36% of patients (bALPL), while nine patients harbored wild-type alleles (wtALPL). Fragility fractures and dental anomalies were more frequent in patients harboring pALPL and bALPL than in wtALPL patients. Of note, wtALPL patients comprised women treated with tamoxifen for hormone-sensitive breast cancer. Mineral and bone markers were similar in the three groups. Mean urine PEA levels were significantly higher in patients harboring pALPL than those detected in patients harboring bALPL and wtALPL; by contrast, serum PLP levels were similar in the three groups. A 6-points score, considering clinical and biochemical features, was predictive of pALPL detection [P = 0.060, OR 1.92 (95% CI 0.972, 3.794)], and more significantly of pALPL or bALPL [P = 0.025, OR 14.33 (95% CI 1.401, 14.605)]. CONCLUSION: In osteopenic/osteoporotic patients, single clinical or biochemical factors did not distinguish hypophosphatasemic patients harboring pALPL or bALPL from those harboring wtALPL. Occurrence of multiple clinical and biochemical features is predictive of ALPL anomalies, and, therefore, they should be carefully identified. Tamoxifen emerged as a hypophosphatasemic drug.

Pyridoxal 5'-phosphate assay based on lucigenin chemiluminescence.

The authors describe the first chemiluminescence (CL) based method for determination of pyridoxal 5'-phosphate (PLP). PLP is found to generate intense CL with lucigenin higher than that of the conventional lucigenin-H2O2 system by a factor of about 9.0. This new finding is used to be in a detection method for PLP via flow injection analysis (FIA). Response is linear in the 50 nM to 200 µM PLP concentration range with a correlation coefficient of 0.998, and the detection limit (at an S/N of 3) is 6.9 nM. The assay is highly selective over various amino acids, vitamins, sugars, coenzymes and metal ions cofactors. It exhibits advantages over the commonly employed HPLC methods in that it is rapid, more economic, eco-friendly and high throughput FIA detection of PLP without the need for toxic derivatization reagents, organic solvents, and HPLC instrumentation. The method was successfully applied to the determination of PLP in (spiked) human blood samples with recoveries in the range from 96.2-101.6% with % RSD < 4.0. The new system is also employed to determine lucigenin in the linear range of 0.3 to 100.0 µM with a correlation coefficient of 0.994 and the limit of detection is 0.04 µM. Graphical abstract Schematic of the chemiluminescent assay for pyridoxal 5'-phosphate (PLP). Lucigenin-PLP demonstrates 9-fold stronger chemiluminescence intensity than the lucigenin-H2O2 system. The detection limit of PLP is 6.9 nM. The method can detect PLP in human serum with good recoveries.

Plasma folate as marker of folate status in epidemiological studies: the European Investigation into Cancer and Nutrition (EPIC)-Potsdam study.

Folate deficiency is often discussed as a potential risk factor for CVD and some cancers. Reliable assessment of folate status in large-scale epidemiological studies is therefore of major importance. The present study assessed the value of plasma folate (PF) compared with erythrocyte folate (EF) as a marker of folate status in 363 participants in the European Investigation into Cancer and Nutrition (EPIC)-Potsdam cohort. EF and PF, total homocysteine (tHcy), pyridoxine, cobalamin, creatinine, total protein and packed cell volume were determined; glutamate carboxypeptidase (GCP) C1561T, reduced folate carrier (RFC) G80A and methylenetetrahydrofolate (MTHFR) C677T polymorphisms were analysed. Anthropometric measurements were taken and dietary intake was assessed with the EPIC-Potsdam food-frequency questionnaire. Comparison of EF and PF with factors that may modulate their concentrations was performed. Cross-classification of blood folates in quintile categories resulted in correct classification into the same or adjacent category of 75.5 % of all subjects. Age, BMI, pyridoxine and cobalamin, fruit and vegetable intake, and vitamin supplementation 24 h before blood draw were positively associated with EF and with PF. For tHcy an inverse association was found. Participants with the MTHFR 677TT genotype showed significantly elevated EF concentrations compared with those with 677CT genotype; EF and PF were more strongly correlated (r 0.78, P<0.0001) for participants with MTHFR 677TT genotype than for those with the 677CC or 677CT genotype. In summary, our present results indicate that plasma folate seems to be a suitable marker for assessment of folate status for use in large-scale epidemiological studies.

Kinetic and structural characterization of Slr0077/SufS, the essential cysteine desulfurase from Synechocystis sp. PCC 6803.

Cysteine desulfurases, designated NifS, IscS, and SufS, cleave L-cysteine to form alanine and an enzyme cysteinyl persulfide intermediate. Genetic studies on the photosynthetic cyanobacterium Synechocystis sp. PCC 6803 have shown that of the three Nif/Isc/SufS-like proteins encoded in its genome only the sequence group II protein, Slr0077/SufS, is essential. This protein has been overexpressed in Escherichia coli, purified to homogeneity, shown to bind pyridoxal-5'-phosphate (PLP) and to catalyze cysteine desulfuration, and characterized in terms of its structure and kinetics. The results suggest that catalysis in the absence of accessory factors has two constituent pathways, one involving nucleophilic attack by C372 to form the Slr0077/SufS-bound cysteinyl persulfide intermediate and the second involving intermolecular attack by the sulfur of a second molecule of the substrate on the initial l-cysteine-PLP complex to form free l-cysteine persulfide. The second pathway is operant in the C372A variant protein, explaining why it retains significant activity, which is proportional to the concentration of l-cysteine (i.e., does not saturate). C-S bond cleavage by the first (normal) pathway is considerably less efficient than the equivalent step in a group I desulfurase (Slr0387) from the same organism (characterized in the accompanying paper). The 1.8 A crystal structure of the protein, which is very similar to that previously reported for E. coli SufS, shows that the loop on which C372 resides is well-ordered and shorter by 11 residues than the corresponding disordered loop of the group I NifS-like protein from Thermotoga maritima. Sequence comparisons establish that the T. maritima and Slr0387 proteins have loops of similar length. The combined structural and kinetic data imply that the modest activity of Slr0077/SufS and other SufS proteins in comparison to their sequence group I (NifS/IscS-like) paralogues results from inefficiency in the nucleophilic attack step associated with differences in the structure or dynamics of this loop. The recent reports that SufS proteins can be activated manyfold by binding to SufE thus implies that the accessory protein either accelerates nucleophilic attack by the conserved cysteine residue of SufS by a conformational mechanism or itself contributes a nucleophilic cysteine for more efficient intermolecular attack.

Effect of physical activity on thiamine, riboflavin, and vitamin B-6 requirements.

Because exercise stresses metabolic pathways that depend on thiamine, riboflavin, and vitamin B-6, the requirements for these vitamins may be increased in athletes and active individuals. Theoretically, exercise could increase the need for these micronutrients in several ways: through decreased absorption of the nutrients; by increased turnover, metabolism, or loss of the nutrients; through biochemical adaptation as a result of training that increases nutrient needs; by an increase in mitochondrial enzymes that require the nutrients; or through an increased need for the nutrients for tissue maintenance and repair. Biochemical evidence of deficiencies in some of these vitamins in active individuals has been reported, but studies examining these issues are limited and equivocal. On the basis of metabolic studies, the riboflavin status of young and older women who exercise moderately (2.5-5 h/wk) appears to be poorer in periods of exercise, dieting, and dieting plus exercise than during control periods. Exercise also increases the loss of vitamin B-6 as 4-pyridoxic acid. These losses are small and concomitant decreases in blood vitamin B-6 measures have not been documented. There are no metabolic studies that have compared thiamine status in active and sedentary persons. Exercise appears to decrease nutrient status even further in active individuals with preexisting marginal vitamin intakes or marginal body stores. Thus, active individuals who restrict their energy intake or make poor dietary choices are at greatest risk for poor thiamine, riboflavin, and vitamin B-6 status.

High expression, purification, and properties of recombinant homocysteine alpha, gamma-lyase.

Homocysteine alpha,gamma-lyase from the anaerobic protozoan parasite Trichomonas vaginalis has been cloned from genomic DNA using PCR methods and expressed in Escherichia coli with a vector containing the T7 promoter. The recombinant homocysteine alpha,gamma-lyase (rHCYase) is expressed as the major protein in the host E. coli cells. The enzyme was purified to approximately 90% purity using heat treatment at 50 degreesC, precipitation steps with polyethyleneimine, polyethylene glycol 8000, and high sodium chloride, DEAE-Sepharose FF chromatography, and phenyl-Sepharose 6 FF chromatography. The final yield was greater than 50%, which encompassed an approximate 18-fold purification. The enzyme is a homotetramer with a monomer molecular weight of 43K and contains pyridoxal phosphate. The Trichomonas rHCYase is selective for homocysteine with respect to very low cysteinase activity in contrast to the alpha,gamma-lyase from Pseudomonas putida, which has very high cysteinase activity with respect to homocysteine. The T. vaginalis and P. putida alpha,gamma-lyases readily separate on a phenyl-Sepharose 6 FF column with the T. vaginalis enzyme eluting first. rHCYase is stable up to 50 degreesC and active over a pH range of 6-8. These properties of high recombinant expression in E. coli, a simple and effective high-yield purification procedure and high relative specificity for homocysteine with respect to cysteine, make rHCYase a promising candidate to use for the diagnosis of hyperhomocystenemia, which has been demonstrated to be a major risk factor for the onset and mortality of cardiovascular disease of all types.

Essential active-site lysine of brain glutamate dehydrogenase isoproteins.

Two soluble forms of bovine brain glutamate dehydrogenase (GDH) isoproteins were inactivated by pyridoxal 5'-phosphate. Spectral evidence is presented to indicate that the inactivation proceeds through Schiff's base formation with amino groups of the enzyme. Sodium borohydride reduction of the pyridoxal 5'-phosphate-inactivated GDH isoproteins produced a stable pyridoxyl enzyme derivative that could not be reactivated by dialysis. The pyridoxyl enzyme was studied through fluorescence spectroscopy. No substrates or coenzymes separately gave complete protection against pyridoxal 5'-phosphate. A combination of 10 mM 2-oxoglutarate with 2 mM NADH, however, gave complete protection against the inactivation. Tryptic peptides of the isoproteins, modified with and without protection, resulted in a selective modification of one lysine. In both GDH isoproteins, the sequences of the peptide containing the phosphopyridoxyllysine were clearly identical to sequences of other GDH species.

Escherichia coli contains a protein that is homologous in function and N-terminal sequence to the protein encoded by the nifS gene of Azotobacter vinelandii and that can participate in the synthesis of the Fe-S cluster of dihydroxy-acid dehydratase.

In this paper, I report the purification of a protein from Escherichia coli that is very similar in sequence, molecular weight, and the reactions it can catalyze to the protein encoded by the Azotobacter vinelandii nifS gene. This E. coli protein contains pyridoxal phosphate as a cofactor and catalyzes the removal of sulfur from cysteine to form alanine and S0. When dithiothreitol is present along with cysteine, the S0 formed is reduced to S2-. This protein has a reactive sulfhydryl group that is essential for activity. As isolated, this sulfhydryl group appears to be in a disulfide linkage with the sulfhydryl group from the phosphopantetheine moiety of the acyl carrier protein. The purified E. coli protein can mobilize the sulfur from cysteine and contribute it to the formation of a [4Fe-4S] cluster on the apoprotein of E. coli dihydroxy-acid dehydratase. A mechanism is proposed for the early stages of the synthesis of Fe-S clusters using this protein and sulfur in the S0 oxidation state.

Glycine decarboxylase: protein chemistry and molecular biology of the major protein in leaf mitochondria.

The four component proteins of the glycine decarboxylase multienzyme complex (the P-, H-, T-, and L-proteins) comprise over one-third of the soluble proteins in mitochondria isolated from the leaves of C3 plants. Together with serine hydroxymethyltransferase, glycine decarboxylase converts glycine to serine and is the site of photorespiratory CO2 and NH3 release. The component proteins of the complex are encoded on nuclear genes with N-terminal presequences that target them to the mitochondria. The isolated complex readily dissociates into its component proteins and reassociates into the intact complex in vitro. Because of the intimate association between photosynthesis and photorespiration, the proteins of the complex are present at higher levels in leaves in the light. The expression of these genes is controlled at the transcriptional level and the kinetics of expression are closely related to those of the small subunit of Rubisco. Deletion analysis of fusions between the promoter of the H-protein of the complex and the reporter gene beta-glucuronidase in transgenic tobacco has identified a region responsible for the tissue specificity and light dependence of gene expression. Gel shift experiments show that a nuclear protein in leaves binds to this region. Glycine decarboxylase has proven to be an excellent system for studying problems in plant biochemistry ranging from protein-protein interactions to control of gene expression.

Molecular studies of glycogen phosphorylase b from bovine liver.

Two active isoforms of bovine liver phosphorylase with distinct subunit composition have previously been purified (Cámara Artigas, A., Barón, C. and Parody-Morreale, A. Prot. Express. Purif. 1994, 5, 157), one showing three SDS-PAGE polypeptide bands (molecular mass = 97, 55 and 40 kDa) and the other showing just one (molecular mass = 97 kDa). A molecular mass of 200 kDa has been determined for the native enzymes by gel filtration. Amino acid analyses have been performed in both cases, giving the same results which are similar to those obtained with other phosphorylases. SDS-PAGE experiments at different concentrations of the three-band enzyme have suggested a 1:1:1 stoichiometry between the polypeptides. The pyridoxal-5'-phosphate site is located in the 55 kDa polypeptide and the phosphorylation site in the 40 kDa one. These polypeptides can be generated from the three-band enzyme by tryptic attack in the presence of glycogen without loss of enzyme activity. In the absence of glycogen, 55 kDa and 38 kDa polypeptides are generated, with a significant decrease in activity. We conclude that the three-band enzyme is a dimer composed of an intact monomer and a broken one. The lyotropic salt activation site of the enzyme is near the amino terminal group.

A fluorometric assay for pyridoxal kinase applicable to crude cell extracts.

We have developed an assay for pyridoxal kinase which takes advantage of the intense fluorescence yield of the oxime of pyridoxal or pyridoxal-5'-phosphate and the substantial difference in the rates of formation of these two oximes. Evidence is presented which demonstrates that the assay is linear with respect to time and amount of protein and is applicable to the activity in crude cell extracts as well as purified enzyme.

Isolation and identification by gas chromatographic mass spectrometry of the carbonyl-active site of pig kidney diamine oxidase.

An adduct with phenylhydrazine was formed with the purified pig kidney diamine oxidase and in parallel with the l-tyrosine decarboxylase from Streptococcus faecalis. The labeled enzymes were hydrolyzed by chemical hydrolysis and the adducts released by hydrolysis were isolated and identified first in HPLC and successively in GC-MS. Both enzymes gave the same adduct which was identified as the phenylhydrazone of pyridoxal. The isolated adduct had the same retention time as the phenylhydrazone of pyridoxal in HPLC and in gas chromatography and showed the same molecular weight in mass spectrometry when chemical ionization was used and the same fragmentation in mass spectrometry when electronic impact was used. The reported results show that pig kidney diamine oxidase contains pyridoxal in the form of covalently linked pyridoxal phosphate which can be released from the enzyme only by chemical hydrolysis. Pig kidney diamine oxidase is therefore a pyridoxal enzyme such as l-tyrosine decarboxylase as hypothesized in the past but never clearly demonstrated.

The regulation of alanine and aspartate aminotransferase by different aminothiols and by vitamin B-6 derivatives.

We examined the effects on alanine aminotransferase and aspartate aminotransferase of different aminothiols (L-cysteine, D-cysteine, cysteamine, L-cysteine ethyl ester, L-cysteine methyl ester) and several vitamin B-6 derivatives (pyridoxal, pyridoxamine, pyridoxol, pyridoxol 5'-phosphate), before and after treatment with KOCN, which transforms these molecules into the corresponding carbamoyl derivatives. Only GPT, and not GOT, was specifically inhibited by L-cysteine and, to a lesser extent, by D-cysteine. The association reaction: PLP + apo GPT<-->holo GPT was inhibited by the vitamin B-6 derivatives, and this inhibition was prevented by pretreatment of the vitamin B-6 derivatives with KOCN. All the observed effects occurred at pH 7, 37 degrees C, at mM and even lower concentrations of reagents. Hence, they all potentially play a physiological role, in the regulation of the PLP dependent enzymes and of the vitamin B-6 levels in the cell.

Studies of the active-site lysyl residue of thermostable aspartate aminotransferase: combination of site-directed mutagenesis and chemical modification.

The gene technological substitution of the cysteinyl residue for the pyridoxal 5'-phosphate-binding lysyl residue (K239) of thermostable aspartate aminotransferase of Bacillus sp. YM-2 led to loss of the activity of the enzyme, which inherently contains no cysteinyl residues. The cysteinyl residue of the mutant enzyme was modified to lysine sulfur analog residues, S-(beta-aminoethyl)cysteine (SAEC), S-(beta-aminopropyl)cysteine (SAPC), and S-(beta-aminoethylthio)cysteine (SATC) with 2-bromoethylamine, 3-bromopropylamine, and 2-mercaptoethylamine, respectively. The modified mutant enzymes showed absorbance at 379 (K239SAEC), 400 (K239SAPC), and 365 nm (K239SATC), whereas the spectrum of the wild-type enzyme exhibited an absorption maximum at 360 nm derived from the internal Schiff base at pH 8.0. The absorption of the modified mutant enzymes at these wavelengths disappeared on reduction with NaCNBH3. This suggests that omega-amino groups of the introduced lysine sulfur analog residue form an internal Schiff base with pyridoxal 5'-phosphate. The modified mutant enzymes showed kcat values of 19.6-0.065% of that of the wild-type enzyme in the overall reaction, and were 10(6)-10(8) times more active than the K239C mutant enzyme. These results suggest that omega-amino groups of the introduced residues of the modified mutant enzyme serve as a catalytic base, and catalysis of the enzyme was affected by the length of the functional side-chain.

L-serine and L-threonine dehydratase from Clostridium propionicum. Two enzymes with different prosthetic groups.

L-Serine dehydratase from the Gram-positive bacterium Peptostreptococcus asaccharolyticus is novel in the group of enzymes deaminating 2-hydroxyamino acids in that it is an iron-sulfur protein and lacks pyridoxal phosphate [Grabowski, R. and Buckel, W. (1991) Eur. J. Biochem. 199, 89-94]. It was proposed that this type of L-serine dehydratase is widespread among bacteria but has escaped intensive characterization due to its oxygen lability. Here, we present evidence that another Gram-positive bacterium, Clostridium propionicum, contains both an iron-sulfur-dependent L-serine dehydratase and a pyridoxal-phosphate-dependent L-threonine dehydratase. These findings support the notion that two independent mechanisms exist for the deamination of 2-hydroxyamino acids. L-Threonine dehydratase was purified 400-fold to apparent homogeneity and revealed as being a tetramer of identical subunits (m = 39 kDa). The purified enzyme exhibited a specific activity of 5 mu kat/mg protein and a Km for L-threonine of 7.7 mM. L-Serine (Km = 380 mM) was also deaminated, the V/Km ratio, however, being 118-fold lower than the one for L-threonine. L-Threonine dehydratase was inactivated by borohydride, hydroxylamine and phenylhydrazine, all known inactivators of pyridoxal-phosphate-containing enzymes. Incubation with NaB3H4 specifically labelled the enzyme. Activity of the phenylhydrazine-inactivated enzyme could be restored by pyridoxal phosphate. L-Serine dehydratase was also purified 400-fold, but its extreme instability did not permit purification to homogeneity. The enzyme was specific for L-serine (Km = 5 mM) and was inhibited by L-cysteine (Ki = 0.5 mM) and D-serine (Ki = 8 mM). Activity was insensitive towards borohydride, hydroxylamine and phenylhydrazine but was rapidly lost upon exposure to air. Fe2+ specifically reactivated the enzyme. L-Serine dehydratase was composed of two different subunits (alpha, m = 30 kDa; beta, m = 26 kDa), their apparent molecular masses being similar to the ones of the two subunits of the iron-sulfur-dependent enzyme from P. asaccharolyticus. Moreover, the N-terminal sequences of the small subunits from these two organisms were found to be 47% identical. In addition, 38% identity with the N-terminus of one of the two L-serine dehydratases of Escherichia coli was detected.

Determination of vitamin B6 vitamers and pyridoxic acid in biological samples.

For the determination of vitamin B6 vitamers (pyridoxal phosphate, pyridoxamine phosphate, pyridoxal, pyridoxine, pyridoxamine) and 4-pyridoxic acid in biological samples such as plasma, cerebrospinal fluid and rat brain regions, a sensitive micromethod using high-performance liquid chromatography (HPLC) with fluorescence detection in combination with post-column derivatization is described. Metaphosphoric acid tissue extracts with deoxypyridoxine as an internal standard were injected into the HPLC system with a binary gradient elution at a flow-rate of 1.2 ml/min. The excitation wavelength of the fluorescence detector was set at 328 nm and the emission wavelength at 393 nm with a 15-nm slit width for the photocell. This method allows the assay of vitamin B6 vitamers within 30 min in one chromatographic run. The present method has been applied extensively for the measurement of vitamin B6 vitamer levels in discrete brain regions of small animals, cells in culture and biopsy samples.

Molecular properties of lysine-2,3-aminomutase.

Lysine-2,3-aminomutase purified from Clostridium subterminale SB4 is reported to exhibit an apparent subunit Mr of 48,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the undenatured enzyme exhibits an apparent Mr of 285,000, as determined by electrophoretic mobility and gel permeation chromatography (Chirpich, T. P., Zappia, V., Costilow, R. N., and Barker, H. A. (1970) J. Biol. Chem. 245, 1778-1789). The diffusion coefficient of the enzyme is 3.36 x 10(-7) cm2/s, as determined by quasielastic light scattering. The overall Mr calculated from the diffusion coefficient and the published sedimentation coefficient is 259,000. Cross-linking experiments using glutaraldehyde and dithiobis(succinimidylpropionate) as cross-linking reagents indicate that the enzyme has a hexameric quaternary structure. The number of major cyanogen bromide peptides, compared with the methionine content of the enzyme, is consistent with the subunits being identical, and isoelectric focusing also is consistent with identical subunits. The circular dichroism of the enzyme indicates that it is a highly ordered structure, which is estimated to consist of 26% alpha-helix and 48% beta-sheet. The enzyme contains approximately six molecules of pyridoxal 5'-phosphate per hexamer, as determined by the phenyl-hydrazine method. The amino acid analysis of the enzyme, after performic acid oxidation, indicates that it contains approximately 13 cysteine residues per subunit. Six sulfhydryl groups per hexamer react readily with 5,5'-dithiobis-2-nitrobenzoate, indicating that one sulfhydryl group is accessible per subunit.

Selenocysteine synthase from Escherichia coli. Nucleotide sequence of the gene (selA) and purification of the protein.

The nucleotide sequence of the selA gene from Escherichia coli whose product is involved in the conversion of seryl-tRNA(Sec UCA) into selenocysteyl-tRNA(Sec UCA) was determined. selA codes for a polypeptide of a calculated Mr of 50,667; a protein of appropriate size was synthesized in vivo in a T7 promoter/polymerase system. An assay for SELA activity was devised which is based on the seryl-tRNA(Sec UCA)-dependent incorporation of [75Se] selenium into acid-insoluble material. It was used to follow SELA purification from cells that overproduced the protein from a phage T7 promoter plasmid. Purified native SELA protein migrates in gel filtration experiments with a native Mr of about 600,000. SELA contains 1 mol of bound pyridoxal 5-phosphate/mol of 50-kDa subunit. Evidence is presented that the overall conversion of seryl-tRNA(Sec UCA) to selenocysteyl-tRNA(Sec UCA) occurs at the SELA protein. SELA, therefore, has the function of a selenocysteine synthase.

Interaction of L-canaline with ornithine aminotransferase of the tobacco hornworm, Manduca sexta (Sphingidae).

Ornithine aminotransferase (L-ornithine:2-oxo-acid aminotransferase (EC 2.6.1.13)) has been purified to homogeneity from last instar larvae of the tobacco hornworm, Manduca sexta (Sphingidae). This enzyme is a 144,000-Da tetramer constructed from 36,000-Da protomeric units. It has a high aspartate/asparagine and glutamate/glutamine content and 2 cysteine residues/subunit. All 8 cysteine residues can react with N-ethylmaleimide to inactivate the enzyme. Maintenance of the enzyme in the presence of 2-mercaptoethanol and dithiothreitol maximizes enzymatic activity and improves storage conditions, presumably by protecting these sulfhydryl groups. The apparent Km values for L-ornithine and 2-oxoglutaric acid are 2.3 and 3.2 mM, respectively. The turnover number is 2.0 +/- 0.1 mumol min-1 mumol-1. L-Canaline (L-2-amino-4-(aminooxy)butyric acid) is a potent ornithine aminotransferase inhibitor. Reaction of the enzyme with L-[U-14C]canaline produces an enzyme-bound, covalently linked, radiolabeled canaline-pyridoxal phosphate oxime. The L-[U-14C]canaline-pyridoxal phosphate oxime has been isolated from canaline-treated enzyme. Dialysis of canaline-inactivated ornithine aminotransferase against free pyridoxal phosphate slowly reactivates the enzyme as the oxime is replaced by pyridoxal phosphate. Analysis of L-[U-14C]canaline binding to ornithine aminotransferase reveals the presence of 4 mol of pyridoxal phosphate/mol of enzyme.

Kinetic studies of the pyridoxal kinase from pig liver: slow-binding inhibition by adenosine tetraphosphopyridoxal.

Pyridoxal kinase from pig liver has been purified 10,000-fold to apparent homogeneity. The enzyme is a dimer of subunits of Mr 32,000. The enzyme is strongly inhibited by the product pyridoxal 5'-phosphate. Liver pyridoxamine phosphate oxidase, another enzyme involved in the biosynthesis of pyridoxal 5'-phosphate, is also strongly inhibited by this compound [Wada, H., & Snell, E. E. (1961) J. Biol. Chem. 236, 2089-2095]. Thus, the biosynthesis of pyridoxal 5'-phosphate in the liver might be regulated by the product inhibition of both pyridoxamine phosphate oxidase and pyridoxal kinase. Kinetic studies revealed that the catalytic reaction of liver pyridoxal kinase follows an ordered mechanism in which pyridoxal and ATP bind to the enzyme and ADP and pyridoxal 5'-phosphate are released from the enzyme, in this order. Adenosine tetraphosphopyridoxal was found to be a slow-binding inhibitor of pyridoxal kinase. Pre-steady-state kinetics of the inhibition revealed that the inhibitor and the enzyme form an initial weak complex prior to the formation of a tighter and slowly reversing complex. The overall inhibition constant was 2.4 microM. ATP markedly protects the enzyme against time-dependent inhibition by the inhibitor, whereas another substrate pyridoxal affords no protection. By contrast, adenosine triphosphopyridoxal is not a slow-binding inhibitor of this enzyme.