Acylation of aspartate aminotransferase

1. Acetylation of aspartate aminotransferase from pig heart inhibits completely the enzymic activity when the coenzyme is in the amino form (pyridoxamine phosphate) or when the coenzyme has been removed, but not when the coenzyme is in the aldehyde form (pyridoxal phosphate). 2. The group the acylation of which is responsible for the inhibition has been identified with the in-amino group of a lysine residue at the coenzyme-binding site. Moreover, in the pyridoxamine-enzyme the amino group of the coenzyme is also acetylated. 3. The reactivity of the coenzyme-binding lysine residue is greatly different in the pyridoxamine-enzyme and in the apoenzyme, suggesting the possibility of an interaction of its in-amino group with pyridoxamine or with other groups on the protein.     

Differential scanning calorimetry of Cd(II) alkaline phosphatases

Differential scanning calorimetry has been employed to monitor structural alterations induced in the dimeric enzyme alkaline phosphatase on binding of Cd(II) (to the metal-free apoenzyme) and phosphate (Pi) (to the Cd(II) enzyme). Cd(II) addition to the apoenzyme at pH 6.5 results in an increased transition temperature, suggesting a stabilizing effect of the bound metal ion. Two distinct structural forms of the protein are detected as discrete calorimetric transitions (Tm = 69-84 degrees C; 87-94 degrees C, respectively). Distribution of the enzyme between these forms is found to depend on the exogenous Cd(II) concentration and the protocol of Cd(II) addition. These results indicate that conversion between the conformational forms is a slow process which appears to require specific levels of metal ion site occupancy. These studies, in which the exogenous Cd(II) concentration was varied from 10(-5) M to 10(-3) M suggest a structural basis for previously observed hysteretic phenomena observed on Cd(II) binding to the enzyme. Even at a minimum stoichiometry of Cd(II) (2 eq/mol of dimer) a single equivalent of Pi is sufficient to accelerate assumption of a stabilized form of the protein (Tm = 90 degrees C). This is followed by a slow structural change paralleling the time course of formation of the functional 2 Cd(II) phosphoryl enzyme which displays two calorimetric transitions (Tm = 65 degrees C, 88 degrees C). The low temperature transition does not appear if Pi is initially present at millimolar concentrations and is abolished on addition of Pi at concentrations in excess of 0.1 mM. These observations suggest the presence of a second, distinct Pi binding site on the 2 Cd(II) phosphoryl enzyme. This is supported by the changes observed in the 31P NMR chemical shift of Pi added to comparable enzyme samples. These data, including assessment of the effect of the presence of Mg(II), are discussed in terms of the mechanism of metal ion association to the enzyme and rearrangement of bound metal ions induced by Pi binding.     

Site-specific methylation of a strategic lysyl residue in aspartate aminotransferase

Conditions for reductive methylation of amine groups in proteins using formaldehyde and cyanoborohydride can be chosen to modify selectively the active site lysyl residue of aspartate aminotransferase among the 19 lysyl residues in each subunit of this protein. Apoenzyme must be treated, under mildly acidic conditions (pH = 6), at a relatively low molar ratio of formaldehyde to protein (40:1); and, upon reduction with sodium cyanoborohydride, 85% of the formaldehyde is incorporated at Lysine 258 and 15% at the amino-terminal alanyl residue. The modified protein, characterized after tryptic hydrolysis, separation of the peptides by high performance liquid chromatography procedures and subsequent amino acid analysis, shows that lysine 258 is preferentially modified as a dimethylated derivative. Modified apoenzyme can accept and tightly bind added coenzyme pyridoxal phosphate, as measured by circular dichroism procedures. The methylated enzyme is essentially catalytically inactive when measured by standard enzymatic assays. On the other hand, addition of the substrate, glutamate, produces the characteristic absorption spectral shifts for conversion of the active site-bound pyridoxal form of the coenzyme (absorbance at 400 nm) to its pyridoxamine form (absorbance at 330 nm). Such a half-transamination-like process occurs as in native enzyme, albeit at several orders of magnitude lower rate. This event takes place even though the characteristic internal holoenzyme Schiff's base between Lys-258 and aldehyde of bound pyridoxal phosphate does not exist in methylated, reconstituted holoenzyme. It is concluded that this chemically transformed enzyme can undergo a half-transamination reaction with conversion of active site-bound coenzyme from a pyridoxal to a pyridoxamine form, even when overall catalytic turnover transamination cannot be detected.     

Studies on the mechanism of acetaldehyde-mediated inhibition of aspartate aminotransferase (GOT) in human erythrocytes

The mechanism of acetaldehyde-mediated GOT inhibition was studied in human red cells. In the GOT assay mix, acetaldehyde competitively inhibits activation of apoGOT by pyridoxal phosphate and pyridoxamine phosphate. However, Ki values are 100-1000 times greater than Km values for these B6 vitamers. Moreover, incubation of undiluted lysates with acetaldehyde at 37 degrees inhibits GOT activity without increasing apoGOT levels and without altering affinity of apoGOT for either B6 coenzyme. In undiluted lysates, inhibition is not prevented by disulfiram. However, incubation at 4 degrees prevents both acetaldehyde metabolism and GOT inhibition while preincubation with NaF prevents GOT inhibition without affecting acetaldehyde disappearance. The effect of NaF is completely reversed by pyruvate but only partially reversed by NADH. Glyceraldehyde 3-phosphate, the only glycolytic intermediate which directly inhibits GOT, does not reverse the NaF effect. Thus, inhibition of GOT by acetaldehyde (a) requires nonoxidative metabolism of acetaldehyde and (b) is not mediated either by glycolytic substrates or by impaired binding of B6 vitamers to the GOT apoenzyme. Since NaF had no effect on a lysate deficient in glucose 6-phosphate dehydrogenase, the hexose monophosphate shunt may play a role in acetaldehyde-mediated GOT inhibition.     

Purification and characterization of aspartate aminotransferase from the thermoacidophilic archaebacterium Sulfolobus solfataricus

Aspartate aminotransferase from the archaebacterium Sulfolobus solfataricus, a thermoacidophilic organism isolated from an acidic hot spring (optimal growth conditions: 87 degrees C, pH 3.5) was purified to homogeneity. The enzyme is a dimer (Mr subunit = 53,000) showing microheterogeneity when submitted to chromatofocusing and/or isoelectric focusing analysis (two main bands having pI = 6.8 and 6.3 were observed). The N-terminal sequence (22 residues) does not show any homology with any stretch of known sequence of aspartate aminotransferases from animal and bacterial sources. The apoenzyme can be reconstituted with pyridoxamine 5'-phosphate and/or pyridoxal 5'-phosphate, each subunit binding 1 mol of coenzyme. The absorption maxima of the pyridoxamine and pyridoxal form are centered at 325 and 335 nm, respectively; the shape of the pyridoxal form band does not change with pH. The enzyme has an optimum temperature higher than 95 degrees C, and at 100 degrees C shows a half-inactivation time of 2 h. The above properties seem to be unique even for enzymes from extreme thermophiles (Daniel, R. M. (1986) in Protein Structure, Folding, and Design (Oxender, D. L., ed) pp. 291-296, Alan R. Liss, Inc., New York) and lead to the conclusion that aspartate aminotransferase from S. solfataricus is one of the most thermophilic and thermostable enzymes so far known.     

Similarity between pyridoxal/pyridoxamine phosphate-dependent enzymes involved in dideoxy and deoxyaminosugar biosynthesis and other pyridoxal phosphate enzymes.

A multiple sequence alignment among aspartate aminotransferase, dialkylglycine decarboxylase, and serine hydroxymethyltransferase (DAS) was used for profile databank search. The DAS profile could detect similarities to other pyridoxal or pyridoxamine phosphate-dependent enzymes, like several gene products involved in dideoxysugar and deoxyaminosugar synthesis. The alignment among DAS and such gene products shows the conservation of aspartate 222 and lysine 258, which, in aspartate aminotransferase, interacts with the N1 of the coenzyme pyridine ring and forms the internal Schiff base, respectively. The lysine is replaced by histidine in the pyridoxamine phosphate-dependent gene products. The alignment indicates also that the region encompassing the coenzyme binding site is the most conserved.     

Determination of pyridoxamine 5'-phosphate in human blood plasma.

Pyridoxamine 5'-phosphate in 18 microliters of human capillary blood plasma is determined by catalytic amplification using the apoenzyme of aspartate aminotransferase. Prior isolation from interfering substances is accomplished by employment of a cation exchange resin in batch operation. The procedure consists of the following stages. Stage I, denaturation of proteins. Trichloroacetic acid is used to precipitate plasma proteins and liberate any bound coenzyme. Dilute NaCl is added to expand the volume thus minimizing coenzyme entrapment in the precipitate. Stage II, isolation of the coenzyme. A sulfonated polystyrene ion exchange resin is used inside a centrifugal filter. Pyridoxamine 5'-phosphate in the supernatant from Stage I adsorbs to the resin. Pyridoxal 5'-phosphate, other organic phosphates, and Pi are removed by centrifugation. Rinsing with dilute NaBH4 destroys traces of pyridoxal 5'-phosphate and washes off residual inhibitors. Pyridoxamine 5'-phosphate is then desorbed with NaOH and Tris buffer and recovered by centrifugation. Stage III, reconstitution and assay. The desorbate from Stage II is incubated with excess apoenzyme. Specific activity of the reconstituted enzyme is measured. Interpolation from a standard curve relating enzyme specific activity and pyridoxamine 5'-phosphate concentration yields the plasma level of the cofactor. Approximately 3 h are required to carry out the procedure. Much of the coenzyme was found not be assayable if plasma was refrigerated overnight or if whole blood was left standing at room temperature for a few hours. The degradation was arrested with freezing at -80 degrees C. In a 13-day experiment involving a healthy subject, sharp rises of plasma pyridoxamine 5'-phosphate were found to occur in response to small doses of oral vitamin B6.(ABSTRACT TRUNCATED AT 250 WORDS)     

The precursor of mitochondrial aspartate aminotransferase is translocated into mitochondria as apoprotein

Mitochondrial aspartate aminotransferase is synthesized on free polysomes as a higher molecular weight precursor (Sonderegger, P., Jaussi, R., Christen, P., and Gehring, H. (1982) J. Biol. Chem. 257, 3339-3345). The present study examines whether the coenzyme pyridoxal phosphate or pyridoxamine phosphate is required for the uptake of the precursor into mitochondria. Chicken embryo fibroblasts were cultured in medium prepared with and without pyridoxal. In cells grown in the presence of pyridoxal only holoform of aspartate aminotransferase and no apoenzyme was detected. Cells cultured under pyridoxal deficiency contained about 30% of apoenzyme in secondary cultures. All of this apoform was identified as mitochondrial isoenzyme. In order to differentiate whether this apoenzyme corresponded to newly synthesized protein or originated from pre-existing holoenzyme, double isotope-labeling experiments were performed. Secondary cultures of chicken embryo fibroblasts grown under pyridoxal depletion were labeled with [3H]methionine, and then pulsed with [35S]methionine. In another series of experiments, the 3H-labeled cells were pulsed with [35S]methionine in the presence of the protonophore carbonyl cyanide m-chlorophenylhydrazone in order to accumulate the precursor. Subsequently, the accumulated precursor was chased into the mitochondria by addition of the carbonyl cyanide m-chlorophenylhydrazone antagonist cysteamine. The holo- and apoenzyme from the ultrasonic extract of the double-labeled cells were separated by affinity chromatography on a phosphopyridoxyl-AH-Sepharose column, immunoprecipitated, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. Under both experimental conditions, the 3H/35S ratio of the apoenzyme was less than half of that of the holoenzyme. Therefore, the apoenzyme and not the holoenzyme is the first product of the precursor in the mitochondria. Apparently, the precursor of mitochondrial aspartate aminotransferase is transported into mitochondria as apoprotein and is processed there independently of the coenzyme.     

Pyridoxal 5'-phosphate and analogs as probes of coenzyme-protein interaction in Baccillus alvei tryptophanase.

Trytophanase from Bacillus alvei was resolved from its coenzyme, pyridoxal phosphate, by treatment with cysteine followed by column chromatography. Spectrophotometric titration of apoenzyme with pyridoxal-P showed 1 mol of pyridoxal-P bound per 52,000 g of enzyme. Kinetic analysis of coenzyme binding showed hyperbolic activation curves with a Km of 1.6 muM. Pyridoxal-P was used as a natural active site probe in spectrophotometric studies to distinguish differences in the active center of holotryptophanase and reconstituted enzyme that were not apparent by other techniques. The pKa for holotryptophanase is 7.9 while the pKa for reconstituted apoenzyme is 8.4. Apotryptophanase binds 2-nor, 2'-methyl, 2'-hydroxy, 6-methyl, and N-oxide pyridoxal-P to form analog enzymes distinguishable on the basis of absorption spectra and relative activity in catalyzing both the alpha, beta-elimination and beta-replacement reactions of tryptophanase. Apoenzyme also binds pyridoxal but pyridoxal analog enzyme is not active.     

Bovine brain low Mr acid phosphatase: purification and properties

Low molecular weight acid phosphatase from bovine brain was purified to homogeneity using affinity chromatography on p-aminobenzylphosphonic acid-agarose to obtain the enzyme with both high specific activity (110 mumol min-1 mg-1 measured at pH 5.5 and 37 degrees C with p-nitrophenyl phosphate as substrate) and good yields. The enzyme was characterized with respect to molecular weight, amino acid composition, pH optimum, Km and Vmax in varying substrates, and to the Ki of varying inhibitors. Furthermore, transphosphorylation to glycerol was demonstrated by measuring the released p-nitrophenol/Pi concentration ratio during the initial phase of the catalyzed reaction. The enzyme was inactivated by iodoacetate and 1,2-cycloexanedione. Inorganic phosphate, a competitive inhibitor, protected the enzyme from being inactivated by the above compounds, demonstrating the involvement of both cysteine(s) and arginine(s) at the active site of the enzyme. Furthermore, the strong inhibition exerted by pyridoxal 5'-phosphate and the low inhibitory capacity possessed by the pyridoxal 5'-phosphate analogues pyridoxamine 5'-phosphate and pyridoxal, indicate that at least one lysine residue is present at the active site.     

Modulation of arginine decarboxylase activity from Mycobacterium smegmatis. Evidence for pyridoxal-5'-phosphate-mediated conformational changes in the enzyme

Arginine decarboxylase (arginine carboxy-lyase, EC 4.1.1.19) from Mycobacterium smegmatis, TMC 1546 has been purified to homogeneity. The enzyme has a molecular mass of 232 kDa and a subunit mass of 58.9 kDa. The enzyme from mycobacteria is totally dependent on pyridoxal 5'-phosphate for its activity at its optimal pH and, unlike that from Escherichia coli, Mg2+ does not play an active role in the enzyme conformation. The enzyme is specific for arginine (Km = 1.6 mM). The holoenzyme is completely resolved in dialysis against hydroxylamine. Reconstitution of the apoenzyme with pyridoxal 5'-phosphate shows sigmoidal binding characteristics at pH 8.4 with a Hill coefficient of 2.77, whereas at pH 6.2 the binding is hyperbolic in nature. The kinetics of reconstitution at pH 8.4 are apparently sigmoidal, indicating the occurrence of two binding types of differing strengths. A low-affinity (Kd = 22.5 microM) binding to apoenzyme at high pyridoxal 5'-phosphate concentrations and a high-affinity (Kd = 3.0 microM) binding to apoenzyme at high pyridoxal 5'-phosphate concentrations. The restoration of full activity occurred in parallel with the tight binding (high affinity) of pyridoxal 5'-phosphate to the apoenzyme. Along with these characteristics, spectral analyses of holoenzyme and apoenzyme at pH 8.4 and pH 6.2 indicate a pH-dependent modulation of coenzyme function. Based on the pH-dependent changes in the polarity of the active-site environment, pyridoxal 5'-phosphate forms different Schiff-base tautomers at pH 8.4 and pH 6.2 with absorption maxima at 415 nm and 333 nm, respectively. These separate forms of Schiff-base confer different catalytic efficiencies to the enzyme.     

Stereochemistry and mechanism of a new single-turnover, half-transamination reaction catalyzed by the tryptophan synthase alpha 2 beta 2 complex

Tryptophan synthase is a versatile enzyme that catalyzes a wide variety of pyridoxal phosphate dependent reactions that are also catalyzed in model systems. These include beta-replacement, beta-elimination, racemization, and transamination reactions. We now show that the apo-alpha 2 beta 2 complex of tryptophan synthase will bind two unnatural substrates, pyridoxamine phosphate and indole-3-pyruvic acid, and will convert them by a single-turnover, half-transamination reaction to pyridoxal phosphate and L-tryptophan, the natural coenzyme and a natural product, respectively. This enzyme-catalyzed reaction is more rapid and more stereospecific than an analogous model reaction. The pro-S 4'-methylene proton of pyridoxamine phosphate is removed during the reaction, and the product is primarily L-tryptophan. We conclude that pyridoxal phosphate enzymes may be able to catalyze some unnatural reactions involving bound reactants and bound coenzyme since the coenzyme itself has the intrinsic ability to promote a variety of reactions.     

Localization of pyrodoxal phosphate binding site on the mero-receptor domain of the glucocorticoid receptor

Previous studies have demonstrated that the vitamin pyridoxal phosphate can alter the physicochemical properties of glucocorticoid receptors. We now report the localization of a pyridoxal phosphate binding site within the mero-receptor domain of this glucocorticoid receptor. Mero-glucocorticoid receptors that are generated by trypsin (10 μg/ml) or chymotrypsin (100 μg/ml) digestion of intact receptors sediment as 2.6 S species on 5–20% sucrose gradients in the presence or absence of pyridoxal phosphate. Mero-glucocoritcoid receptors prepared by exogenous proteinases are hydrophobic and show no affinity for DEAE Bio-Gel A. Treating either trypsin-generated or chymotrypsin-generated mero-receptors with pyridoxal phosphate rapidly converts the proteins (60 and 35%, respectively) into forms that bind to DEAE Bio-Gel A. Induction of DEAE binding is specific to pyridoxal phosphate, for treating mero-receptors with pyridoxal, pyridoxamine or pyridoxine phosphate is ineffective. Furthermore, DEAE binding cannot be induced by adding other pyridoxal phosphate-treated cytosols to untreated mero-receptors. High-resolution polyacrylamide gel isoelectric focussing studies indicated that treating mero-receptor generated by either proteinase with pyridoxal phosphate shifted the isoelectric points of lower pH values. The conversion of the mero-receptor to a more acidic form also occurred when the intact glucocorticoid receptor was treated with the vitamin prior to proteolysis. These studies localize at least one pyridoxal phosphate binding site on the mero-receptor domain of the rat thymocyte glucocorticoid receptor.     

Observations on the pyridoxal 5'-phosphate inhibition of DNA polymerases.

Pyridoxal 5'-phosphate at concentrations greater than 0.5 mM inhibits polymerization of deoxynucleoside triphosphate catalyzed by a variety of DNA polymerases. The requirement for a phosphate as well as aldehyde moiety of pyridoxal phosphate for inhibition to occur is clearly shown by the fact that neither pyridoxal nor pyridoxamine phosphate are effective inhibitors. Since the addition of nonenzyme protein or increasing the amount of template primer exerted no protective effect, there appears to be specific affinity between pyridoxal phosphate and polymerase protein. The deoxynucleoside triphosphates, however, could reverse the inhibition. The binding of pyridoxal 5'-phosphate to enzyme appears to be mediated through classical Schiff base formation between the pyridoxal phosphate and the free amino group(s) present at the active site of the polymerase protein. Kinetic studies indicate that inhibition by pyridoxal phosphate is competitive with respect to substrate deoxynucleoside triphosphate(s).     

31P nuclear-magnetic-resonance studies of pyridoxal and pyridoxamine phosphates. Interaction with cytoplasmic aspartate transaminase.

The 31P nuclear magnetic resonance (NMR) spectrum of the phosphate in free pyridoxal or pyridoxamine phosphate reveals a resonance signal that is coupled to the methylene protons of the 5-CH2 with JHP of 6.0 +/- 0.3 Hz. Proton noise decoupling results in a single signal with a pH-dependent chemical shift with deprotonation of the phosphate resulting in a shift of the 31P resonance to lower fields. A single 31P NMR signal at a frequency corresponding to fully ionized phosphate monoesters is observed in aspartate-transaminase-bound pyridoxal or pyridoxamine phosphate. The 31P resonance in the holotransaminase is pH-independent and is unaffected by saturating concentrations of substrates or inhibitors. Only denaturation with 6 M guanidine with HCl results in changes in the 31P of the holoenzyme. It appears that the phosphate group of pyridoxal phosphate is bound to a positive pocket in the holoenzyme and remains fully ionized in the pH range of 5.6 to 9.2. The phosphate-binding properties are present even in the apoenzyme which is able to bind inorganic phosphate which then can be displaced by pyridoxal or pyridoxamine phosphate in the process of holoenzyme formation.     

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-oxoglutarate 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.     

The binding of the coenzyme pyridoxal 5''-phosphate and analogues of the substrate-coenzyme complex to tyrosine decarboxylase.

Phosphopyridoxyl derivatives, which are stable analogues of a substrate-coenzyme complex, are bound at the active site with great affinity. From a comparison of the interaction of a number of such compounds with the apoenzyme the delta G0 values for the binding of the substrate carboxy and phenyl groups and of the coenzyme aldehydic group were determined to be equal to (or more negative than) -3.8. -8.4 and -12.5kJ/mol (-0.9, -1.9 and -3kcal/mol) respectively; the delta G0 for the binding of the coenzyme phosphate group was shown to be more negative than -20.5kJ/mol (-4.9kcal/mol). Two features of the binding process of the coenzyme-substrate analogues to tyrosine decarboxylase have already been found in the case of tyrosine aminotransferase [Borri-Voltattorni, Orlacchio, Giartosio, Conti & Turano (1975) Eur. J. Biochem. 53, 151-160]: (1) in the binding of the substrate to the enzyme a significant fraction of the instrinsic delta G0 appears to be used for some associated endoergonic process; (2) the delta H0 and delta S0 of binding appear to be very sensitive indicators of the correct alignment of the substrate-coenzyme and analogues at the active site.     

On the inhibitory activity of 4-vinyl analogues of pyridoxal: enzyme and cell culture studies.

Analogues of pyridoxal and of pyridoxal phosphate in which the 4-CHO group is replaced with CH = CH2 were synthesized and were found to be potent inhibitors of pyridoxal kinase and pyridoxine phosphate oxidase of rat liver. They also inhibited the growth of mouse Sarcoma 180 and mammary adenocarcinoma TA3 in cell culture. Saturation of the vinyl double bond, replacement of the 5-CH2OH with methyl, methylation of the phenolic hydroxyl, or conversion to the N-oxide resulted in diminution or loss of all these activities. Similarly, the introduction of a beta-methyl group into the vinyl analogues of pyridoxal reduced all these inhibitory activities. The 4-vinyl anatogue of pyridoxal was shown to be a substrate of pyridoxal kinase and the product a potent inhibitor of pyridoxine oxidase, competing with pyridoxal phosphate. The affinity of this phosphorylated pyridoxal analogue to some apoenzymes varied greatly, indicating striking differences among the cofactor binding sites of these enzymes. The growth inhibitory effects of these analogues on cells in culture correlated well with their effects on pyridoxal kinase and pyridoxine phosphate oxidase in cell-free systems.     

Study of the reorientation of coenzyme in active sites of aspartate-aminotransferase isoenzymes using linear dichroism method

Cytosolic and mitochondrial pig aspartate aminotransferases (cAAT and mAAT) and chicken cAAT were oriented in a compressed slab of polyacrylamide gel. Linear dichroism (LD) spectra of the pyridoxal and pyridoxamine forms of AATs and of complexes of the pyridoxal form with substrate analogues have been recorded. The tilt angles of the coenzyme at the intermediary steps of the transamination reaction have been calculated on the basis of reduced LD values (delta A/A), atomic coordinates of the coenzyme and directions of the transition dipole moments in the coenzyme ring. It was assumed that rotation of the coenzyme ring occurs around the C2-C5 axis in all cases except the enzyme complex with glutarate: in the latter case the direction N1-C4 was assumed to be a rotation axis. It has been found that formation of the enzyme complex with glutarate and protonation of the internal aldimine induce dissimilar reorientations of the coenzyme. As a result of protonation, the coenzyme tilts by 27 degrees in cAAT and 13 degrees in mAAT. Formation of the external aldimine with 2-methylaspartate is accompanied by tilting of the coenzyme ring by 44 degrees in cAAT and 39 degrees in mAAT. For the quinonoid complex with erythro-3-hydroxyaspartate, the tilt angles were found to be 63 degrees in cAAT and 53 degrees in mAAT. It was inferred that the basic features of the active site dynamics are similar in three AATs studied. The differences in the coenzyme tilt angles between cAAT and mAAT might be linked to catalytic peculiarities of the isoenzymes.     

Activation of Glutamate Apodecarboxylase by Succinic Semialdehyde and Pyridoxamine 5''-Phosphate

Glutamate apodecarboxylase was activated by incubation with succinic semialdehyde and pyridoxamine 5'-phosphate. Activation required both compounds and was highly selective for succinic semialdehyde. Of 18 analogs tested, only glyoxylate, pyruvate, oxaloacetate, and 2-oxoglutarate activated the apoenzyme significantly, but much higher concentrations of these compounds than of succinic semialdehyde were required. In the presence of pyridoxamine 5'-phosphate, the concentration of succinic semialdehyde giving half-maximal activation of apoenzyme was 7 microM. In contrast, the Ki for succinic semialdehyde as a competitive inhibitor of glutamate decarboxylation was 1.2 mM, indicating that apoenzyme with bound pyridoxamine 5'-phosphate has a much higher affinity for succinic semialdehyde than does holoenzyme. The concentration of pyridoxamine 5'-phosphate giving half-maximal activation was 17 microM, which is more than an order of magnitude greater than the corresponding value for pyridoxal 5'-phosphate.