Pyridoxamine Phosphate Form Research Articles

Reversible modification of amino groups in aspartate aminotransferase

Amino groups in the pyridoxal phosphate, pyridoxamine phosphate, and apo forms of pig heart cytoplasmic aspartate aminotransferase ( l-aspartate:2-oxglutarate aminotransferase, EC 2.6.1.1) have been reversibly modified with 2.4-pentanedione. The rate of modification has been measured spectrophotometrically by observing the formation of the enamine produced and this rate has been compared with the rate of loss of catalytic activity for all three forms of the enzyme. Of the 21 amino groups per 46 500 molecular weight, approx. 16 can be modified in the pyridoxal phosphate form with less than a 50% change in the catalytic activity of the enzyme. A slow inactivation occurs which is probably due to reaction of 2,4-pentanedione with the enzyme-bound pyridoxal phosphate. The pyridoxamine phosphate enzyme is completely inactivated by reaction with 2,4-pentanedione. The inactivation of the pyridoxamine phosphate enzyme is not inhibited by substrate analogs. A single lysine residue in the apoenzyme reacts approx. 100 times faster with 2,4-pentanedione than do other amino groups. This lysine is believed to be lysine-258, which forms a Schiff base with pyridoxal phosphate in the holoenzyme

Fluorine-19 nuclear magnetic resonance studies of effects of ligands on trifluoroacetonylated supernatant aspartate transaminase.

The selective reaction of Cys-45 and -82, on the one hand, and Cys-390, on the other, with 3-bromo-1,1,1-trifluoropropanone allows for the probing of these regions of aspartate transaminase in the absence and in the presence of enzymatic ligands by 19F nuclear magnetic resonance (NMR). The 19F chemical shifts of the resonance lines differ for the three cysteines and so does their behavior with pH changes. The resonance signals with chemical shifts at 615 and 800 Hz upfield from trifluoroacetic acid correspond to modified cysteine-82 and -45 and have tentatively been assigned in this order. The 615-Hz resonance is affected by pH changes that fit best the influence of a single ionizing residue. On the 800-Hz line, the pH changes appear to be the influence of a minimum of two ionizing residues. The 19F resonance from modified Cys-390 is pH independent in the pH range 5-9 for the pyridoxal phosphate, pyridoxamine phosphate, and apoenzyme forms of the enzyme. Occupation of the active site by a quasi-enzyme-substrate complex, trifluoromethionine pyridoxyl phosphate, affects the 19F chemical shift of modified Cys-390, making it pH dependent with a pK value of 8.4. The 19F NMR properties of the pyridoxal form of Cys-390-modified enzyme can be used to monitor some ligand interactions with the active-center region. Addition of alpha-ketoglutarate or succinate to the ketone labeled enzyme causes a decrease in the resonance line width, and titrations show that this procedure is a good method with which to study the affinity of the enzyme for these ligands. The interpretation of the chemical shift and line-width characteristics of the 19F resonance arising from Cys-390 are most consistent with a model in which the region around this residue seems to be affected by conformational changes arising from substrate binding to the active-center subsites in productive (covalent) manner. Nonproductive complexes which possess fast ligand-protein exchange, such as those between alpha-ketoglutarate or succinate with the pyridoxal phosphate form of the enzyme, may result only in a greater degree of freedom for Cys-390.

Carbamylation of aspartate transaminase and the pK value of the active site lysyl residue.

Abnormal lysyl residues can be detected in aspartate transaminase by following the rate of reaction of amino groups with KN14CO and the rate of enzymatic inactivation. Peptide isolation subsequent to carbamylation of the apoenzyme produces a peptide which is absent in the carbamylated holoenzyme. The composition of the carbamylated peptide matches that of a tryptic peptide containing the active site Lys-258. The holoenzyme retains full catalytic activity after carbamylation of its NH2-terminal alanine and lysyl residues other than Lys-258, which is protected by aldimine formation with pyridoxal phosphate. Apoenzyme prepared from KNCO-treated holoenzyme (apoenzyme') is susceptible to further carbamylation at Lys-258 with irreversible loss of catalytic activity. Carbamylation of the active site lysyl residue is 25 to 50 times more rapid than that of the other 18 lysyl residues of aspartate transaminase. The kinetics of inactivation by KNCO at different pH values served to determine the pH-independent second order rate constant (k) and the pK of the amino group of Lys-258. These values are pK = 7.98 +/- 0.08 and k = 146 +/- 5 M-1S-1, which are similar to the values determined for carbamylation of the NH2- terminal groups of human hemoglobin (Garner, M. H., Bogardt, R. A., and Gurd, E. R. N. (1975) J. Biol. Chem. 250, 4398-4404). The pK value for Lys-258 is as low as that for a group in the active site region which can perturb a 19F nuclear magnetic resonance probe inserted into that region (Martinez-Carrion, M., Slebe, J. C., Boettcher, B., and Relimpio, A. M. (1976) J. Biol. Chem. 251, 1853-1858). Apoenzyme carbamylated at Lys-258 can accept pyridoxal phosphate at the active site even though no Schiff base in formed. Furthermore, this active site carbamylated holoenzyme will form spectroscopically detectable enzyme-substrate complexes with amino acids. The complexes slowly convert to species with absorbance identical with that of enzyme in the pyridoxamine phosphate form.

Interactions between Glutamate Dehydrogenase, Transaminases, and Keto Acids

Data are presented which are consistent with the concept that transaminases can be bound to glutamate dehydrogenase. In the presence of TPNH and ammonium ions the transaminase in this enzyme-enzyme complex can be converted to the pyridoxamine phosphate form. Keto acids bind to and react with the transaminase in this complex to yield the pyridoxal phosphate form of the transaminase, TPN, and the corresponding amino acid. The specific structure of the keto acid required for binding to the enzyme-enzyme complex is determined by the transaminase, and not glutamate dehydrogenase. For example, oxalacetate is bound better than pyruvate to the complex of glutamate-oxalacetate transaminase and glutamate dehydrogenase while the opposite is the case when glutamate-pyruvate transaminase is part of the complex. The conversion of oxalacetate to aspartate occurs more readily with the complex between glutamate-oxalacetate transaminase and glutamate dehydrogenase than with the latter enzyme alone. With the enzyme-enzyme complex the specific activity with respect to glutamate dehydrogenase is higher (in some cases over 70-fold); the concentration of oxalacetate required to produce maximal activity is more than 1000-fold lower, and the concentration of ammonium ion required to produce maximal activity is more than 10-fold lower. Also, in the presence of the enzyme-enzyme complex, ADP is a significant activator. The activity of the complex can be regulated by several factors. For example, the specific activity and activation by ADP decreases when the concentration of glutamate dehydrogenase exceeds about 0.1 mg per ml. If the concentrations of keto acid and glutamate dehydrogenase are both high, then the reaction is similar to that of the keto acid with glutamate dehydrogenase alone. If the concentrations of the transaminase and glutamate dehydrogenase are both high, the pyridoxamine phosphate form of the transaminase is produced faster than the capacity of the keto acid to react and TPN is produced in excess of the amino acid.

Physical-Chemical Studies on the Pyridoxal Phosphate Binding Site in Sodium Borohydride-reduced and Native Phosphorylase

Abstract NaBH4-reduced phosphorylase b is a catalytically active species which retains many of the properties of the native enzyme. One property that is strikingly different, however, is the spectral behavior of bound pyridoxal phosphate. At pH 7.0, the pyridoxal phosphate shows essentially no absorption at 333 nm, where pyridoxal phsophate in the native enzyme absorbs; absorption at 333 nm returns upon lowering the pH. The 333 nm band at low pH in the reduced enzyme was found to be optically active, (ΔA/A) 333 nm ≅ 0.7 x 10-3. Circular dichroism of the reduced phosphorylase b at pH 7.0 indicated that no new band was formed above 300 nm; the ultraviolet spectrum also provided no evidence for a band above 300 nm. Guanidine·HCl (4.25 m) and sodium dodecyl sulfate (0.3%) caused the appearance of a band near 330 nm at pH 6.8. Difference spectra of reduced phosphorylase b (±sodium dodecyl sulfate; pH 5 versus pH 7) indicated that the hidden band must be below 300 nm. AMP (0.01 m) was shown to cause partial reversal of the hidden band to 333 nm conversion at pH 6.0; ATP was also effective. Substrates and AMP had no effect on the spectrum of the reduced phosphorylase b at pH 6.8. Ultracentrifugal studies showed that native and NaBH4-reduced enzyme have different conformations. The data were interpreted to mean that the 333 nm and hidden band forms of NaBH4-reduced phosphorylase represent different enzyme conformations. The hidden band form is thought to represent a form of pyridoxal phosphate in which the pyridinium nitrogen is unprotonated and the 3-hydroxyl group undissociated or strongly hydrogen bonded; the 333 nm species is believed to be a dipolar form of pyridoxal phosphate which is exposed to solvent. Spectral properties of enzyme-bound pyridoxal phosphate in native phosphorylase were compared to NaBH4-reduced enzyme, and it was suggested that pyridoxal phosphate exists in these two forms at pH 7.0 in the same microscopic environment. Circular dichroism of native phosphorylase b at 422 nm was observed. The presence of phosphate (0.01 m) and AMP (0.001 m) did not perturb the observed long wave length circular dichroism. Solvent perturbation studies showed that enzyme-bound pyridoxal phosphate in native phosphorylase is not available to ethylene glycol. From our results, evaluation of model compound data, and fluorescence results, a new structure was proposed for the 333 nm form for native phosphorylase. This species is thought to be a neutral tautomeric imine of pyridoxal phosphate.

The uptake of pyridoxal phosphate by human red blood cells.

In order to examine whether Pyridoxal phosphate could or could not be transferred into red blood cells in the form of a phosphate ester, the red blood cells were incubated with 14C-32P-pyridoxal phosphate and the following results were obtained.1. The radioactivity of the extracellular medium decreased with time, and the same amount of radioactivity was found in the cell fraction. The ratio of 14C:32P in the medium or in the cells did not change throughout the reaction. 2. Most of the radioactivities present in the cells were recovered in the form of pyridoxal phosphate. The ratio of 14C:32P of pyridoxal phosphate in the cells was the same as that in the medium. A slight amount of radioactivity of 14C of pyridoxal and 32P of the inorganic phosphate were observed in the cells. 3. NaF and CH2ICOOH inhibited the transfer of pyridoxal phosphate to the extent of 50%. At low temperature, the transfer of the radioactivity into the cell was remarkably depressed. 4. After hemolysis, the radioactivity in the red blood cells was liberated in the supernatant. By starch-gel electrophoresis and Sephadex-gel filtration, pyridoxal phosphate was found to be combined with hemoglobin. 5. The red blood cells containing pyridoxal phosphate was incubated with hemoglobin solution, but pyridoxal phosphate was not liberated from the cells. The pyridoxal phosphate was not hydrolyzed by alkaline phosphatase. These results suggested that pyridoxal phosphate was not merely adsorbed on the surface of cell membrane, but was transferred through the cell membrane. These observations lead to the conclusion that pyridoxal phosphate was transferred into the red blood cells without being hydrolyzed to pyridoxal and inorganic phosphate.

Studies of gluconeogenic mitochondrial enzymes: V. The effect of transaminases on reactions catalyzed by glutamate dehydrogenase

Transaminases (the glutamate-oxaloacetate from either bovine liver mitochondria or pig heart and the glutamate-pyruvate from pig heart) are activators of reactions catalyzed by glutamate dehydrogenase. Unlike activation by many ligands as steroids and purine nucleotides, transaminase activates reactions with both monoand dicarboxylic substrates and mainly activates by decreasing the Michaelis constant for ammonium ions. The intact transaminase with pyridoxal phosphate is required for activation. There is much less activation when the concentration of glutamate dehydrogenase is low (reaction with α-ketoglutarate) or in the presence of GTP than when enzyme concentration is high (reaction with pyruvate or oxaloacetate) and in the presence of ADP. Other results are consistent with the concept that glutamate dehydrogenase has transaminase dehydrogenase activity. That is in the presence of TPNH, ammonium chloride and glutamate-oxaloacetate transaminase, glutamate dehydrogenase catalyzes the conversion of the transaminase in the pyridoxal phosphate form to the pyridoxamine phosphate form. Keto-acids can rapidly react with the pyridoxamine form of the transaminase producing the pyridoxal form plus an amino acid. Therefore, in the presence of keto-acids the overall reaction is an amino acid dehydrogenase. This is apparently why transaminases activate reactions catalyzed by glutamate dehydrogenase. The transaminase is not an activator but in the presence of transaminase there are two amino acid dehydrogenase reactions. The transaminase dehydrogenase reaction is significant only in the presence of high concentrations of both enzymes and when α-ketoglutarate and glutamate are either absent or present in low concentrations. It seems that pyridoxal phosphate on the transaminase is bound to the α-ketoglutarate site of glutamate dehydrogenase. Also the transaminase is probably preferentially bound to polymeric forms of glutamate dehydrogenase. Some possible physiological implications of the transaminase dehydrogenase reaction are discussed. This reaction could be of some physiological value when the concentration of amino acids are too low to convert a significant amount of the transaminase into the pyridoxamine form.