Rabbit liver pyridoxamine (pyridoxine) 5'-phosphate oxidase. Purification and properties.

Pyridoxamine (pyridoxine) 5'-phosphate oxidase (EC 1.4.3.5) has been purified 2000-fold from rabbit liver. The enzyme preparation migrates as a single protein and activity band on analytical disc gels containing 4,7, or 9 percent acrylamide, and as a single protein band on sodium dodecyl sulfate acrylamide gels. The oxidase is, therefore, homogeneous by these criteria. The pure enzyme catalyzes the following reactions in the presence of FMN: (See journal for formula). These activities copurify in the ratio of 1:1:1. Apparent K-m values are 10 muM for pyridoxamine-P, 30 muM for pyridoxine-P, and 40 nM for FMN. Apparent K-m values for N-(phosphopyridoxyl)amines range from 3.1 times 10-5 M to 1.6 times 10-3 M. The dissociation constant for FMN binding, determined by quenching of protein fluorescence, is 20 nM. The pH optima for all three types of substrates are broad, with maxima near pH 9. The pH dependence of FMN binding, measured by quenching of flavin fluorescence, has the same shape as the substrate activity profile. The holoenzyme has absorption maxima red-shifted from those of FMN to 380 nm and 448 nm, and exhibits spectral changes typical of flavoproteins upon reduction with dithionite. Its oxidation-reduction potential at pH 7 in phosphate buffer is -0.131 volt. The native enzyme has a molecular weight of 54,000 and is made up of two possibly identical polypeptide chains with molecular weights of 27,000. The applicability of proposed mechanisms of flavin catalysis to this flavoprotein is discussed.     

Activation and inactivation of rabbit liver pyridoxamine (pyridoxine) 5'-phosphate oxidase activity by urea and other solutes.

Urea, guanidine hydrochloride, and neutral salts both activate and denature pyridoxamine (pyridoxine) 5′-phosphate oxidase (EC 1.4.3.5) from rabbit liver. Activation occurs at lower concentrations (e.g. 2-2.5 m for urea) of these compounds and is rapid and reversible. Greater structural changes leading to inactivation occur slowly under “activating conditions” but rapidly at higher concentrations of urea. Both reversibly and irreversibly inactivated species are formed. Activation by urea does not involve either dissociation of the enzyme to subunits or aggregation to multimers, and there is little disruption of protein secondary structure. The V and K_m for substrates, K_i for product, and the rate of release of product from the enzyme are increased by urea, and substrate inhibition is decreased; urea has little effect on the reactivity of reduced enzyme with oxygen. Both flavin and tryptophanyl fluorescence increase in the presence of urea; at lower concentrations of urea (≤2 m), there is a rapid increase followed by slower, sigmoidal increases. The polarization of flavin fluorescence of the oxidase is increased upon the addition of 2 m urea, which corresponds to the initial enhancement of protein and flavin fluorescence intensities, and then decreases. The near-ultraviolet-visible absorption spectra of native enzyme and that treated with 2 m urea are only slightly different; however, a considerable change at the flavin-binding site is reflected by the circular dichroism spectra. Hence, it appears that urea yields a rapidly formed, “activated” species of the oxidase that is changed primarily at the active site in a manner that allows increased dissociation of substrate and product.     

Further evidence for an essential histidyl residue at the active site of pig liver 5-aminolevulinic acid dehydratase

Photoxidation with methylene blue and rose bengal and chemical modification by diethylpryrocarbonate of pig liver 5-aminolevulinic acid dehydratase produced strong inactivation of the enzyme which was concentration dependent. Loss of enzyme activity by both photoxidation and ethoxyformylation was pH and time-dependent and protected by the presence of the substate and competitive inhibitors. The rate of inactivation was directly related to the state of protonation of histidyl groups, the unprotonated from being modified at a much faster rate than the protonated form. Plots of the pseudo-first order rate constants for 5-aminolevulinic acid dehydratase inactivation against pH resulted in typical titration curves showing inflection points at about pH 6.4 for methylene blue and rose bengal and 6.8 for diethylprocarbonate providing further and unequivocal evidence for the existence of critical histidyl groups at the active centre of the enzyme.     

N-(5'-phospho-4'-pyridoxyl)amines as substrates for pyridoxine (pyridoxamine) 5'-phosphate oxidase

A preparation of pyridoxine (pyridoxamine) 5′-phosphate oxidase, with a specific activity of 9,400 nmoles/hr/mg protein, 10-fold higher than that previously reported, was used to study the oxidation of various N-(5′-phospho-4′-pyridoxyl)amines. Values for K_m, from 3.1 × 10^?5 M to 1.6 × 10^?3 M, and for V_max, relative to pyridoxamine-P, of 20 to 140% were obtained. Compounds lacking a 5′-phosphate were not substrates, and the enzymic reaction was dependent on the presence of both FMN and O₂. N-(phosphopyridoxyl)-L-amino acids had lower K_m's than the corresponding -D-amino acid compounds. When 1-¹⁴C-N-(phosphopyridoxyl)glycine was used as a substrate, no ¹⁴CO₂ was evolved, and 1-¹⁴C-glycine was detected in the incubation mixture.     

Spectroscopic studies of pyridoxamine (pyridoxine) 5'-phosphate oxidase. Equilibrium dissociation constants and spectra for riboflavin 5'-phosphate and analogues.

Pyridoxamine (pyridoxine) 5'-phosphate oxidase (EC 1.4.3.5) has been shown to bind 1 mol of riboflavin 5'-phosphate (FMN) per mol of apoenzyme and is active with or inhibited by numerous FMN analogues [Kazarinoff, M. N., & McCormick, D. B. (1975) J. Biol. Chem. 250, 3436--3442]. The KD values and spectra for selected apoenzyme--flavin complexes have been determined and used to elucidate some of the properties of the FMN-binding site of this flavoprotein. Alterations of the pyrimidinoid portion of the flavin ring decrease binding considerably. The absorption spectra for the protein complexes with 3-deaza-FMN and 8-hydroxy-FMN indicate the presence of a dipolar or positively charged protein group near N1 and O2. The substitution of methyl for hydrogen at N3 apparently causes distortion of the interaction between the flavin ring and an active-site aromatic amino acid residue. Although binding is also decreased somewhat by substitutions at postions 8 and 8 alpha, considerable bulk [e.g., 8-(diethylamino)-FMN and 8 alpha-S-(N-acetyl-cysteinyl)-FMN] is accommodated. Hence, this portion of the flavin ring is probably oriented toward, possibly in contact with, solvent, as has been found for the flavodoxins. The importance of optimum interactions between the flavin and the apoprotein is further emphasized by large differences in the activity of flavin analogues that have similar midpoint potentials in solution.     

Evidence for an essential arginine residue at the active site of ATP citrate lyase from rat liver.

Rat liver ATP citrate lyase was inactivated by 2, 3-butanedione and phenylglyoxal. Phenylglyoxal caused the most rapid and complete inactivation of enzyme activity in 4-(2-hydroxyethyl)-1-piperazine-ethanesulphonic acid buffer, pH 8. Inactivation by both butanedione and phenylglyoxal was concentration-dependent and followed pseudo- first-order kinetics. Phenylglyoxal also decreased autophosphorylation (catalytic phosphate) of ATP citrate lyase. Inactivation by phenylglyoxal and butanedione was due to the modification of enzyme arginine residues: the modified enzyme failed to bind to CoA-agarose. The V declined as a function of inactivation, but the Km values were unaltered. The substrates, CoASH and CoASH plus citrate, protected the enzyme significantly against inactivation, but ATP provided little protection. Inactivation with excess reagent modified about eight arginine residues per monomer of enzyme. Citrate, CoASH and ATP protected two to three arginine residues from modification by phenylglyoxal. Analysis of the data by statistical methods suggested that the inactivation was due to modification of one essential arginine residue per monomer of lyase, which was modified 1.5 times more rapidly than were the other arginine residues. Our results suggest that this essential arginine residue is at the CoASH binding site.     

Evidence for one essential tryptophan residue at the active site of relaxin.

The hormone relaxin, which is responsible for the rapid widening of the birth channel in mammals prior to parturition, was purified from hog ovarian extracts and shown to be homogeneous by exclusion chromatography in 6 M guanidine hydrochloride and SDS gel electrophoresis. Of the two disulfide-linked chains that comprise relaxin, the larger chain was shown to contain two tryptophan residues, one of which could be completely oxidized in native relaxin without measurable effect on its biological activity. Oxidation of the second residue completely inactivated the hormone. Modifications of lysine side chains or carboxymethylation of a single methionine residue at low pH did not impair the effectiveness of relaxin.     

A radiometric assay of pyridoxamine (pyridoxine) 5′-phosphate oxidase

A radiometric assay for pyridoxamine 5′-phosphate oxidase (pyridoxamine (pyridoxine) 5′-phosphate:O₂ oxidoreductase (deaminating), EC 1.4.3.5) has been developed utilizing N-(5′-phosphopyridoxyl)[³H]tryptamine. This assay is more sensitive than previously used colorimetric and fluorescent assays for this oxidase and furthermore is applicable to erythrocytes. Tritiated substrate is incubated with an enzyme sample in the presence of excess unlabeled truptamine and the radiolabeled tryptamine product is extracted into toluene and quantitated by liquid scintillation counting.     

Evidence for an essential arginine residue at the active site of Escherichia coli acetate kinase

Escherichia coli acetate kinase (ATP: acetate phosphotransferase, EC 2.7.2.1.) was inactivated in the presence of either 2,3-butanedione in borate buffer or phenylglyoxal in triethanolamine buffer. When incubated with 9.4 mM phenylglyoxal or 5.1 mM butanedione, the enzyme lost its activity with an apparent rate constant of inactivation of 0.079 min-1, respectively. The loss of enzymatic activity was concomitant with the loss of an arginine residue per active site. Phosphorylated substrates of acetate kinase, ATP, ADP and acetylphosphate as well as AMP markedly decreased the rate of inactivation by both phenylglyoxal and butanedione. Acetate neither provided any protection nor affected the protection rendered by the adenine nucleotides. However, it interfered with the protection afforded by acetylphosphate. These data suggest that an arginine residue is located at the active site of acetate kinase and is essential for its catalytic activity, probably as a binding site for the negatively charged phosphate group of the substrates.     

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...     

Evidence for the essential histidine residue at the active site of glucose/xylose isomerase from Streptomyces

Modification of glucose/xylose isomerase from Streptomyces sp. NCIM 2730 by diethylpyrocarbonate (DEPC) or its photo-oxidation in presence of rose bengal or methylene blue caused rapid loss in its activity. The inactivation of the enzyme was accompanied by an increase in the absorbance at 240 nm and was reversed by hydroxylamine. Glucose and xylose but not Mg++ and Co++ afforded significant protection to the enzyme from inactivation by DEPC. Inactivation followed pseudo-first-order kinetics and modification of a single histidine residue per mole of enzyme was indicated.     

X-ray structure of Escherichia coli pyridoxine 5'-phosphate oxidase complexed with pyridoxal 5'-phosphate at 2.0 A resolution.

Escherichia coli pyridoxine 5'-phosphate oxidase catalyzes the terminal step in the biosynthesis of pyridoxal 5'-phosphate by the FMN oxidation of pyridoxine 5'-phosphate forming FMNH(2) and H(2)O(2). Recent studies have shown that in addition to the active site, pyridoxine 5'-phosphate oxidase contains a non-catalytic site that binds pyridoxal 5'-phosphate tightly. The crystal structure of pyridoxine 5'-phosphate oxidase from E. coli with one or two molecules of pyridoxal 5'-phosphate bound to each monomer has been determined to 2.0 A resolution. One of the pyridoxal 5'-phosphate molecules is clearly bound at the active site with the aldehyde at C4' of pyridoxal 5'-phosphate near N5 of the bound FMN. A protein conformational change has occurred that partially closes the active site. The orientation of the bound pyridoxal 5'-phosphate suggests that the enzyme catalyzes a hydride ion transfer between C4' of pyridoxal 5'-phosphate and N5 of FMN. When the crystals are soaked with excess pyridoxal 5'-phosphate an additional molecule of this cofactor is also bound about 11 A from the active site. A possible tunnel exists between the two sites so that pyridoxal 5'-phosphate formed at the active site may transfer to the non-catalytic site without passing though the solvent.     

Histidine as an essential residue in the active site of the copper enzyme galactose oxidase.

The pH dependence of the oxidation of β-methyl-d-galactopyranoside by galactose oxidase at 1.33 mm O₂ has been determined. The k_cat exhibits a bell-shaped dependence on the ionization of at least two groups in the enzyme-substrate complex, pK_b' = 6.3 and pK_a' = 7.1, respectively. The pH-independent value for k_cat at 1.33 mm O₂ (nonsaturating) and saturating glycoside is 1435 s^?; the pH optimum is 6.7. Galactose oxidase is inactivated rapidly by iodoacetamide. Although the reaction is much slower, iodoacetate also inactivates the enzyme. The inactivation by iodoacetamide obeys saturation kinetics; at pH 7.0 k₃ = 2.19 min^?1 and K_i = 5.1 mM; k₃ but not K_i exhibits a bell-shaped pH dependence, with pK_a values of 6.3 and 7.6, respectively. Labeling with [¹⁴C]iodoacetamide establishes that one carboxamidomethyl group is incorporated per enzyme molecule. This incorporation parallels the loss of enzymatic activity. Only N-3-carboxymethylhistidine is detected in chromatograms following hydrolysis of the labeled protein. The protein-bound copper is not lost as a consequence of alkylation. Apogalactose oxidase does not react with iodoacetamide. The alkylation is inhibited by the oxidation of an active center tryptophan residue (s) by N-bromosuccinimide. The fraction of residual enzyme activity remaining after tryptophan oxidation corresponds to the extent of labeling by [¹⁴C]iodoacetamide. Although alkylation causes little change in the spin Hamiltonian parameters of the Cu(II) atom, it nearly abolishes both the optical activity and optical absorbance of the metal. The native tryptophan fluorescence of the enzyme, which is a sensitive probe of its active site, is also markedly affected. Since binding of a substrate, β-methyl-d-galactopyranoside, reduces fluorescence as it does in the active enzyme and binding of CN^? at the Cu(II) site as detected by electron spin resonance appears unaffected by the alkylation, the effect of alkylation is on catalysis, per se. Both a catalytic and a subtle conformational role for the active site histidine are inferred from the results.     

Evidence of an essential histidine residue in rabbit liver aryl sulfatase A.

A detailed study of the pH dependence of the Michaelis-Menten constants (V and K_m) of aryl sulfatase A (EC 3.1.6.1) from rabbit liver indicates that at least two functional groups (pK's ~4.3 and ~7 in the enzyme-substrate complex) participate in the enzymic degradation of substrate. Aryl sulfatase A is inactivated by diethyl pyrocarbonate (ethoxyformic anhydride). The enzyme that has been modified with this reagent can in turn be reactivated by treatment with hydroxylamine. The pH dependence of inactivation reveals a reactive group having a pK of 6.5–7.0. The results indicate that at least one histidine plays an important catalytic role in rabbit liver aryl sulfatase A, consistent with the results of earlier workers who employed diazotized sulfanilic acid. Phosphate ion, a competitive inhibitor, partially protects the enzyme from inactivation by diethyl pyrocarbonate whereas sulfate ion, also a competitive inhibitor, increases the rate of inactivation by diethyl pyrocarbonate. This result is of particular significance in view of the anomalous kinetics of aryl sulfatase A. The kinetic effects of even small amounts of sulfate ion impurities in many commercial sulfate ester substrate preparations is also discussed.     

An essential residue at the active site of aspartate transcarbamylase.

Reaction of phenylglyoxal with aspartate transcarbamylase and its isolated catalytic subunit results in complete loss of enzymatic activity. This modification reaction is markedly influenced by pH and is partially reversible upon dialysis. Carbamyl phosphate or carbamyl phosphate with succinate partially protect the catalytic subunit and the native enzyme from inactivation by phenylglyoxal. In the native enzyme complete protection from inactivation is afforded by N-(phosphonacetyl)-L-aspartate. The decrease in enzymatic activity correlates with the modification of 6 arginine residues on each aspartate transcarbamylase molecule, i.e. 1 arginine per catalytic site. The data suggest that the essential arginine is involved in the binding of carbamyl phosphate to the enzyme. Reaction of the single thiol on the catalytic chain with 2-chloromercuri-4-nitrophenol does not prevent subsequent reaction with phenylglyoxal. If N-(phosphonacetyl)-L-aspartate is used to protect the active site we find that phenylglyoxal also causes the loss of activation of ATP and inhibition by CTP. The rate of loss of heterotropic effects is exactly the same for both nucleotides indicating that the two opposite regulatory effects originate at the same location on the enzyme, or are transmitted by the same mechanism between the subunits, or both.