Studies on the biosynthesis of cyclitols, XXXVII. On mechanism and function of Schiff's base formation as an intermediary reaction step of myo-inositol-1-phosphate synthase from rat testicles.

It could be shown that the formation of a Schiff's base by myo-inositol-1-phosphate synthase of rat testicles occurs by binding the aldehyde group of the open form of its substrate, D-glucose 6-phosphate, to a lysyl residue of one or both smaller subunits of the enzyme. The participation of the Schiff's base formation in the catalytic process is supported by the observations that (a) no Schiff's base is formed if NAD is removed from the enzyme, and (b) in the presence of NAD, the dehydrogenation step involved in the catalytic mechanism apparently takes place rapidly after the formation of the Schiff's base.     

Dissection of the early steps in the porphobilinogen synthase catalyzed reaction. Requirements for Schiff's base formation

The porphobilinogen (PBG) synthase catalyzed reaction requires both Zn(II) and reducing equivalents for the production of PBG from two molecules of 5-aminolevulinic acid (ALA). An early step in the reaction is the production of a Schiff's base between PBG synthase and one ALA molecule. Because both substrate molecules are chemically identical, there had been no evidence of enzyme-catalyzed partial reactions of ALA under conditions where PBG is not formed. In this study, NaBH4 was used to trap the Schiff's base formed between substrate ALA and active holo-PBG synthase, inactive apo-PBG synthase, and inactive methylmethanethiosulfonate-modified apo-PBG synthase. ALA-dependent NaBH4 inactivation of these enzyme forms was quantified at 50-62, 94-97, and 93-96% inactivation, respectively. [4-14C]ALA was used to determine the stoichiometry of Schiff's base trapping which was 2.3, 3.5-4.0, and 3.4 per octamer for holoenzyme, apoenzyme, and methylmethanethiosulfonate-modified apoenzyme, respectively. These results are consistent with four active sites per octamer or half-of-the-sites reactivity. We conclude that the production of the Schiff's base formed between one ALA molecule and the enzyme requires neither Zn(II) nor reduced enzyme sulfhydryl groups. Furthermore, the possible number of kinetic schemes for formation of the quaternary complex of enzyme, Zn(II), and two ALA moieties, one as the Schiff's base, has been reduced from 12 to 3. This is the first demonstration of a partial reaction catalyzed by PBG synthase with the natural substrate ALA under conditions which do not support PBG formation. Thus, we have opened the way toward investigating the partial reactions which may precede Zn(II) participation in the PBG synthase reaction.     

Possible involvement of Lys603 from Escherichia coli glucosamine-6-phosphate synthase in the binding of its substrate fructose 6-phosphate

Pyridoxal 5'-phosphate is a competitive inhibitor of glucosamine-6-phosphate synthase with respect to the substrate fructose 6-phosphate. Irreversible inactivation of pyridoxal-5'-phosphate-treated enzyme with [14C]-cyanide resulted in covalent incorporation of close to 1 mol pyridoxal 5'-phosphate/mol enzyme subunit. The enzyme-pyridoxal-5'-phosphate complex could also be inactivated by reduction with NaBH3CN. Sequence analysis of the unique radioactively labelled tryptic peptide, resulting from inactivation with [3H]NaBH3CN, identified the C-terminal nonapeptide encompassing the modified Lys603. The presence of fructose 6-phosphate protected this residue from pyridoxylation. Direct evidence that a lysine residue is involved in the binding of the substrate as a Schiff base came from the isolation at 4 degrees C of a enzyme-fructose-6-phosphate complex in a 1:1 molar ratio. Treatment of the enzyme-[14C]fructose-6-phosphate complex with NaBH3CN revealed one site of modification in the tryptic peptide map. In contrast, trapping the same complex with potassium cyanide resulted in the isolation of several radiolabelled peptides containing lysines which could potentially bind fructose 6-phosphate. However, since the radioactivity was not specifically associated with the lysine residues, it is suggested that these 14C-labelled peptides resulted from the decomposition of an unstable alpha,alpha'-dihydroxyaminonitrile adduct rather than from a lack of specificity of fructose 6-phosphate fixation. Lys603 is then the candidate of choice for fructose 6-phosphate binding since it lies at or near the active site as demonstrated by the trapping experiments with pyridoxal 5'-phosphate described above, and among the lysines which belong to the sugar-binding domain this is the only one conserved between the three members of the purF, glutamine-dependent, amidotransferase subfamily which include the glucosamine-6-phosphate synthase from Escherichia coli, Saccharomyces cerevisiae and the Rhizobium nodulation protein NodM.     

Studies on the biosynthesis of cyclitols, XXXVI. Purification of myo-inositol-1-phosphate synthase of the duckweed, Lemna gibba, to homogeneity by affinity chromatography on NAD-Sepharose molecular and catalytic properties of the enzyme.

Using the technique of affinity chromatography on NAD-Sepharose the myo-inositol-1-phosphate synthase of Lemna gibba was purified to homogeneity. The molecular and catalytic properties of this enzyme differ very much from those of myo-inositol-1-phosphate synthase from animal sources. Thus the specific activity of the duckweed enzyme is more than two orders of magnitude higher than that of the enzyme from rat testes. It is inhibitied by EDTA and can be reactivated by Mn2. Its molecular weight (135000 +/- 5000), its subunit composition (3 subunits with identical electrophoretic behaviour) and its isoelectric point (pH 7.7) are also very different from the corresponding parameters for the animal enzyme.     

Inactivation of brain myo-inositol monophosphate phosphatase by pyridoxal-5'-phosphate

Myo-inositol monophosphate phosphatase (IMPP) is a key enzyme in the phosphoinositide cell-signaling system. This study found that incubating the IMPP from a porcine brain with pyridoxal-5'-phosphate (PLP) resulted in a time-dependent enzymatic inactivation. Spectral evidence showed that the inactivation proceeds via the formation of a Schiff's base with the amino groups of the enzyme. After the sodium borohydride reduction of the inactivated enzyme, it was observed that 1.8 mol phosphopyridoxyl residues per mole of the enzyme dimer were incorporated. The substrate, myo-inositol-1-phosphate, protected the enzyme against inactivation by PLP. After tryptic digestion of the enzyme modified with PLP, a radioactive peptide absorbing at 210 nm was isolated by reverse-phase HPLC. Amino acid sequencing of the peptide identified a portion of the PLP-binding site as being the region containing the sequence L-Q-V-S-Q-Q-E-D-I-T-X, where X indicates that phenylthiohydantoin amino acid could not be assigned. However, the result of amino acid composition of the peptide indicated that the missing residue could be designated as a phosphopyridoxyl lysine. This suggests that the catalytic function of IMPP is modulated by the binding of PLP to a specific lysyl residue at or near its substrate-binding site of the protein.     

Immunohistochemical Staining and Enzyme Activity Measurements Show myo-Inositol-1-Phosphate Synthase to Be Localized in the Vasculature of Brain

Rabbit anti-bovine myo-inositol-1-phosphate synthase was used to examine the distribution of that enzyme in perfused and immersion-fixed bovine brain and testis. In brain, intense and specific staining was found in the walls of all the vascular elements including cerebral capillaries. The remainder of brain parenchyma exhibited only low levels of background staining. In testis, an organ rich in the enzyme, blood vessels showed no specific staining. Instead, the enzyme was found in the seminiferous epithelium of the seminiferous tubules, perhaps localized in spermatozoa. To confirm the brain finding, the activity of myo-inositol-1-phosphate synthase was measured in bovine brain microvessel preparations and brain pial vessels. In these preparations the activity of the enzyme was found on average to be 7 and 22 times enriched over that in whole brain, respectively. The activities of two other enzymes of inositol metabolism, myo-inosose reductase and myo-inositol-1-phosphatase, were also examined for their distribution in brain. Those enzymes were found to be generally distributed. The surprising finding of a vascular localization of myo-inositol-1-phosphate synthase in brain raises new questions about the mechanism by which myo-inositol is concentrated to such high cellular levels in the principal substance of that organ.     

Separation of Neurospora crassa myo-inositol-1-phosphate synthase from glucose-6-phosphate dehydrogenase by affinity chromatography

The purification of Neurospora crassa myo-inositol-1-phosphate synthase (EC 5.5.1.4) was studied by affinity chromatography using the substrate (glucose-6-phosphate), the inhibitor (pyrophosphate), the coenzyme (NAD+) and the coenzyme analogues (5'AMP and Cibacron Blue F3G-A) of the enzyme as adsorbents attached to agarose gel. Myo-inositol-1-phosphate synthase could be separated completely from the contaminating substance, glucose-6-phosphate dehydrogenase (EC 1.1.1.49), on Blue Sepharose CL-6B and on pyrophosphate-Sepharose. The purified enzyme had a specific activity of 16 400 U/mg. The sodium dodecyl sulfate/polyacrylamide gel electrophoresis of the 60 micrograms of this purified enzyme gave a homogenous band. The enzyme was found to be composed of four identical subunits having a molecular weight of 65 000.     

Separation of Neurospora Crassa Myo-Inositol-1-Phosphate Synthase from Glucose-6-Phosphate Dehydrogenase by Affinity Chromatography

The purification of Neurospora crassa myo-inositol-1-phosphate synthase (EC 5.5.1.4) was studied by affinity chromatography using the substrate (glucose-6-phosphate), the inhibitor (pyrophosphate), the coenzyme (NAD⁺) and the coenzyme analogues (5′AMP and Cibacron Blue F3G-A) of the enzyme as adsorbents attached to agarose gel. Myo-inositol-1-phosphate synthase could be separated completely from the contaminating substance, glucose-6-phosphate dehydrogenase (EC 1.1.1.49), on Blue Sepharose CL-6B and on pyrophosphate-Sepharose. The purified enzyme had a specific activity of 16 400 U/mg. The sodium dodecyl sulfate/polyacrylamide gel electrophoresis of 60 μq of this purified enzyme gave a homogenous band. The enzyme was found to be composed of four identical subunits having a molecular weight of 65 000.     

The regulation of myo-inositol-1-phosphate-synthase activity from Neurospora crassa by pyrophosphate and some cations.

The effect of several cations on the inhibition by PPi of the enzyme myo-inositol-1-phosphate synthase (1L-myo-inositol-1-phosphate lyase (isomerizing) EC 5.5.1.4) from Neurospora crassa was studied. The study wastol biosynthesis can occur in the presence of an intracellular PPi concentration which exceeds the Ki for the enzyme by 350-fold. The inhibition of enzyme activity by PPi,at pH 7.7, was reversed, in decreasing order of effectiveness, by Mn-2, Fe-3     

A chromogenic substrate for phosphatidylinositol-specific phospholipase C: 4-nitrophenyl myo-inositol-1-phosphate.

A chromogenic water-soluble substrate for phosphatidylinositol-specific phospholipase C was synthesized starting from myo-inositol employing isopropylidene and 4-methoxytetrahydropyranyl protecting groups. In this analogue of phosphatidylinositol, 4-nitrophenol replaces the diacylglycerol moiety, resulting in synthetic, racemic 4-nitrophenyl myo-inositol-1-phosphate. Using this synthetic substrate a rapid, convenient and sensitive spectrophotometric assay for the phosphatidylinositol-specific phospholipase C from Bacillus cereus was developed. Initial rates of the cleavage of the nitrophenol substrate were linear with time and the amount of enzyme used. At pH 7.0, specific activities for the B. cereus enzyme were 77 and 150 mumol substrate cleaved min-1 (mg protein)-1 at substrate concentrations of 1 and 2 mM, respectively. Under these conditions, less than 50 ng quantities of enzyme were easily detected. The chromogenic substrate was stable during long term storage (6 months) as a solid at -20 degrees C.     

Mechanism of action of uridine diphoglucose dehydrogenase. Evidence for an essential lysine residue at the active site

The oxidation of UDP-glucose by the enzyme UDP-glucose dehydrogenase (EC 1.1.1.22) from beef liver has been shown to proceed via the enzyme-bound intermediate, UDP-alpha-D-glyco-hexodialdose. The enzyme does not release this aldehyde, nor can it be trapped by reaction with hydroxylamine, thiosemicarbazide, or cyanide. Tight binding of the intermediate aldehyde can be explained by the recent observation that the essential thiol group of the enzyme forms a thiohemiacetal with the aldehyde during the course of the reaction. However, an enzyme preparation with the essential thiol derivatized with cyanide will still not release the aldehyde, indicating an additional as yet unknown binding mechanism. Derivatization ([14C]formaldehyde, followed by NaBH4 reduction) of 6 of the approximately 168 lysine residues per enzyme molecule (of six catalytic subunits) results in destruction of 47% of the enzyme activity, suggesting the involvement of an essential reactive lysine in the mechanism. Preincubation of the enzyme with UDP-glucose decreases both the loss of activity and incorporation of the label, indicating that this lysine is in the vicinity of the active site. Acid hydrolysis of the labeled preparation, followed by paper chromatography, shows that the label has a mobility, in the system used, that is identical with lysine. Elution of this spot followed by chromatography on Aminex A-5 resin showed that it contained the expected mixture of epsilon-N-methyl lysines. When enzyme that has its essential thiol derivatized with cyanide is incubated with UDP-[14C]glucose and NAD+, and then reduced with NaB3H4, a stable enzyme complex is formed which contains both labels. Acid hydrolysis of this preparation, followed by either two-dimensional paper chromatography or separation in an amino acid analyzer, results in both labels appearing in the position of lysine. It is evident that the enzyme oxidizes the UDP-[14C]glucose to the corresponding aldehyde which occurs as the Schiff's base with an essential lysine. This is then reduced by the NaB3H4 to form a secondary amine which is stable toward hydrolysis and migrates with lysine in separation procedures. As would be predicted, the enzyme can be similarly labeled by treatment with UDP-alpha-D-gluco-hexodisidose alone, followed by NaB3H4 reduction. The same hydrolysis product results from this procedure, and it behaves identically with the product formed by treating alpha-N-acetyl lysine with UDP-alpha-D-gluco-hexodialdose, reducing with NaBH4, and then hydrolyzing. This substance appears to be N5-((5-formyl-2-furanyl)methyl)lysine. When chromatographed on Aminex A-5, both the model compound and enzyme hydrolysate gave peaks corresponding to free lysine and the proposed derivative. Evidence is presented that the oxidation of UDP-glucose to the aldehyde is a concerted reaction involving the formation of the Schiff's base, rather than the formation of the aldehyde with the subsequent formation of the Schiff's base...     

Modification of glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides with the 2',3'-dialdehyde derivative of NADP+ (oNADP+)

Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides is irreversibly inactivated by the 2,3'-dialdehyde of NADP+ (oNADP+) in the absence of substrate. The inactivation is first order with respect to NADP+ concentration and follows saturation kinetics, indicating that the enzyme initially forms a reversible complex with the inhibitor followed by covalent modification (KI = 1.8 mM). NADP+ and NAD+ protect the enzyme from inactivation by oNADP+. The pK of inactivation is 8.1. oNADP+ is an effective coenzyme in assays of glucose-6-phosphate dehydrogenase (Km = 200 microM). Kinetic evidence and binding studies with [14C] oNADP+ indicate that one molecule of oNADP+ binds per subunit of glucose-6-phosphate dehydrogenase when the enzyme is completely inactivated. The interaction between oNADP+ and the enzyme does not generate a Schiff's base, or a conjugated Schiff's base, but the data are consistent with the formation of a dihydroxymorpholino derivative.     

Characterization of recombinant Drosophila melanogaster myo-inositol-1-phosphate synthase expressed in Escherichia coli

Cloned myo-inositol-1-phpsphate synthase (INOS) of Drosophila melanogaster was expressed in Escherichia coli, and purified using a His-affinity column. The purified INOS required NAD+ for the conversion of glucose-6-phosphate to inositol-1-phosphate. The optimum pH for myo-inositol-1-phosphate synthase is 7.5, and the maximum activity was measured at 40 degrees C. The molecular weight of the native enzyme, as determined by gel filtration, was approximately Mr 271,000 +/- 15,000. A single subunit of approximately Mr 62,000 +/- 5,000 was detected upon SDS-polyacrylamide gel electrophoresis. The Michaelis (Km) and dissociation constants for glucose-6-phosphate were 3.5 and 3.7 mM, whereas for the cofactor NAD+ these were 0.42 and 0.4 mM, respectively.     

A fluorescent substrate for the continuous assay of phosphatidylinositol-specific phospholipase C: synthesis and application of 2-naphthyl myo-inositol-1-phosphate.

A fluorescent water-soluble substrate for phosphatidylinositol-specific phospholipase C was synthesized. The diacylglycerol moiety of the natural substrate, phosphatidylinositol, was replaced by the fluorescent moiety, 2-naphthol, resulting in the synthetic substrate, racemic 2-naphthyl myo-inositol-1-phosphate. The synthetic substrate provided a continuous fluorometric assay for the phosphatidylinositol-specific phospholipase C from Bacillus cereus. Initial rates of the cleavage of the 2-naphthyl substrate by the phospholipase measured by fluorometry were linear with time and the amount of enzyme added. The specific enzyme activity at pH 8.5 and 25 degrees C was about 0.04 mumol/min mg protein at an initial substrate concentration of 0.8 mM. 31P NMR experiments suggest that, as with phosphatidylinositol itself, cleavage of the fluorescent substrate proceeds in two steps via a myo-inositol-1,2-cyclic phosphate intermediate, and that only the D-isomer is a substrate for the B. cereus phospholipase. The synthetic substrate was stable during long-term storage as a solid in the dark at -20 degrees C. It was also stable for several weeks when stored in the dark frozen in aqueous solution near neutral pH.     

Characterization of the binding of the anti-sickling compound, BW12C, to haemoglobin.

The anti-sickling agent BW12C [Beddell, Goodford, Kneen, White, Wilkinson & Wootton (1984) Br. J. Pharmacol. 82, 397-407] was designed to left-shift the oxygen saturation curve of haemoglobin (HbA) by preferential binding to the oxy conformation at a single site between the terminal amino groups of the alpha-chains through Schiff's base formation, ionic and hydrophobic interactions. In the present work, Schiff's base linkages formed with [14C]BW12C were reduced with NaBH4 and the alpha- and beta-globin chains separated. Under oxy conditions at a molar ratio of 2:1, the covalently bound BW12C is localized almost exclusively on a single alpha-chain; tryptic digestion confirms the terminal amino group (alpha 1-valine) as the reaction site, in accord with the design hypothesis. However, about half the labelled BW12C is released on tetramer disruption, suggesting the presence of additional non-covalent binding. Under deoxy conditions, alpha- and beta-chains are labelled approximately equally, and at higher molar ratios additional binding in both oxy and deoxy conditions is seen. Isoelectric-focusing studies under oxy conditions show a complex pattern of modified bands for both HbA and HbA1c (blocked beta-terminal amino groups) but no modification for HbA carbamylated at both alpha- and beta-terminal amino groups or at the alpha-chains only, again confirming the alpha-terminal amino region as the main interaction site. Equilibrium dialysis measurements under oxy conditions indicate two strong binding sites with a binding constant of less than 10(-6) M and a number of weaker binding sites. The present data thus confirm that BW12C binds at the intended locus but reveal additional non-covalent binding at an undefined site, and weaker binding through Schiff's base formation with other amino groups.     

Mechanism of action of 5-aminolaevulinate dehydratase from human erythrocytes.

Purified 5-aminolaevulinate dehydratase (porphobilinogen synthase, EC 4.2.1.24) from human erythrocytes was incubated initially with limiting amounts of 5-amino [5-14C]laevulinate in a rapid-mixing apparatus. The single-turnover reaction with respect to the bound labelled 5-aminolaevulinate was completed by the addition of unlabelled 5-aminolaevulinate and the resulting radioactive porphobilinogen was isolated and degraded. The 14C label was found to be located predominantly at C-2 of the product, demonstrating that, of the two substrate molecules participating in the reaction, the 5-aminolaevulinate molecule initially bound to the enzyme provides the propionic acid 'side' of the porphobilinogen. The same enzyme-[14C]substrate species that yields regiospecific porphobilinogen may be trapped by reaction with NaBH4, showing that the substrate molecule initially bound to the enzyme does so in the form of a Schiff base. A conventional incubation with 5-amino[5-14C]laevulinate yielded porphobilinogen with an equal distribution of the label between C-2 and C-11. The reaction mechanism of the human erythrocyte 5-aminolaevulinate dehydratase thus follows the same course as that of other dehydratases studied in our laboratory by using single-turnover techniques.     

Evidence for the existence of a novel enzyme system. myo-Inositol-1-phosphate dehydrogenase in Phaseolus aureus.

A novel enzyme system, myo-inositol-1-phosphate dehydrogenase, has been isolated from germinating mung bean seeds. The dehydrogenation and cleavage of myo-inositol 1-phosphate by this enzyme leads to the synthesis of a pentose phosphate which appears to be ribulose 5-phosphate. The pH optimum of the enzyme is 8.6; NAD+ is required as coenzyme and no other nucleotides can replace NAD+. Mono- or divalent cations are not essential for the enzyme activity. Stoichiometry of the reaction suggests that 2 mol of NAD+ are reduced per mol of ribulose-5-P generated.     

Electron spin resonance and nuclear relaxation studies on spin-labeled glutamate dehydrogenase.

The reaction of glutamate dehydrogenase with two different stable nitroxides (spin labels) is reported. The two compounds contain a carbonyl and an iodoacetamide group as their reactive parts. The carbonyl compound inactivates the enzyme by the formation of a 1:1 covalent complex after NaBH4 reduction of an intermediate Schiff's base. Evidence indicates that the enzyme is modified at lysine-126 in the active site. The electron spin resonance (ESR) spectrum of spin-labeled enzyme indicates a high degree of immobilization of the nitroxide. The binding of reduced coenzyme NADPH is reflected by a change (immobilization) of the ESR spectrum. Nuclear relaxation of bound substrate, oxidized coenzyme, and inhibitor by the paramagnetic group is observed. This shows the existence of a binding site for these compounds close to the active site. The distances of selected protons of the binding ligands to the nitroxide are calculated. The iodoacetamide spin label reacts with several groups, one of which is not a sulfhydryl. The reaction of this particular group causes inactivation of the enzyme. Protection against this inactivation could be achieved with certain ligands. Only enzyme that was spin labeled without such protection caused paramagnetic relaxation of bound substrate and coenzyme.