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.

pH dependence of the kinetic parameters of L-asparaginase.

The concentration dependence of the rate of hydrolysis of L-asparagine by Escherichia coli L-asparaginase (L-asparagine amidohydrolase, EC 3.5.1.1) has been measured over the range pH 4.5 to pH 9.1 by a direct spectrophotometric assay at 220 nm and by a coupled assay utilizing glutamate dehydrogenase to detect the ammonia produced. The velocity of the hydrolysis reaction at saturating levels of substrate is independent of pH over this interval. The plot of V/km over the same interval is bell-shaped, being dependent on pKa values of 6.58 and 8.69. The higher pKa is attributed to the amino group of asparagine. The lower pKa is associated with the enzyme active site and is probably due to an imidazole group.

Isolation and sequence of an active-site peptide from maize leaf phosphoenolpyruvate carboxylase inactivated by pyridoxal 5'-phosphate.

An active-site peptide from maize (Zea mays L.) phosphoenolpyruvate carboxylase has been isolated, sequenced and identified in the primary structure following chemical modification/inactivation of the enzyme by pyridoxal 5'-phosphate and reduction with sodium borohydride. The amino acid sequence of the purified dodecapeptide is Val-Gly-Tyr-Ser-Asp-Ser-Gly-L*ys-Asp-Ala-Gly-Arg, which corresponds exactly to residues 599-610 in the deduced primary sequence of the maize-leaf enzyme. Comparative analysis of the deduced amino acid sequences of the enzyme from Escherichia coli, Anacystis nidulans and C3, C4 and Crassulacean acid metabolism plants indicates that they all contain this specific lysyl group, as well as a high degree of sequence homology flanking this species-invariant residue. This observation suggests a critical role for Lys-606 during catalysis by maize phosphoenolpyruvate carboxylase. This represents the first identification of a specific, species-invariant active-site residue in the enzyme.

Phosphoenol-3-bromopyruvate. A mechanism-based inhibitor of phosphoenolpyruvate carboxylase from maize.

Phosphoenol-3-bromopyruvate is an excellent competitive inhibitor (versus phosphoenolpyruvate) of maize phosphoenolpyruvate carboxylase (Ki = 30 microM), provided that preincubation of enzyme with inhibitor is avoided. If the enzyme is preincubated with inhibitor in the presence of Mn2+ and HCO3(-), complete inactivation occurs over the course of about 1 h. The inactivation is first order in enzyme and is saturable with respect to inhibitor, with an apparent Michaelis constant of 67.5 microM and a half-life at high inhibitor concentration of 13.4 min. The inactivation is inhibited by phospholactate and is much slower at low HCO3(-) concentration. Incubation of the enzyme with phosphoenol-3-bromopyruvate in the presence of H14CO3(-) and Mn2+ followed by treatment with NaBH4 leads to incorporation of 14C into the inactive enzyme. If treatment with NaBH4 is omitted, the enzyme is inactivated, but no 14C is incorporated into the inactive product. It appears that phosphoenol-3-bromopyruvate is a mechanism-based inactivator of phosphoenolpyruvate carboxylase, probably because of enzyme-catalyzed conversion to 3-bromooxalacetate, which alkylates the enzyme.

Carbon isotope effect on dehydration of bicarbonate ion catalyzed by carbonic anhydrase.

The carbon-13 kinetic isotope effect on the dehydration of HCO3- by bovine carbonic anhydrase has been measured. To accomplish this, bicarbonate was added to a buffer solution at pH 8 containing carbonic anhydrase under conditions where purging of the product CO2 from the solution is rapid. Measurement of the isotopic composition of the purged CO2 as a function of the concentration of carbonic anhydrase permits calculation of the isotope effect on the enzymic reaction. The isotope effect on the dehydration is k12/k13 = 1.0101 +/- 0.0004. This effect is most consistent with a ping-pong mechanism for carbonic anhydrase action, in which proton transfer to or from the enzyme occurs in a step separate from the dehydration step. Substrate and product dissociation steps are at least 2-3-fold faster than the hydration/dehydration step.

(Z)-3-(fluoromethyl)phosphoenolpyruvate: synthesis and enzymatic studies.

(Z)-3-(Fluoromethyl)phosphoenolpyruvate has been synthesized in nine chemical steps from glyoxylic acid. The compound is stable at pH 3, but at pH 8 it decomposes within seconds to give 2-oxo-3-butenoate. When 3-(fluoromethyl)phosphoenolpyruvate is added to a solution of phosphoenolpyruvate carboxylase or pyruvate kinase, the enzyme is inactivated over the course of an hour. Identical kinetics of inactivation are observed whether the reaction is initiated by addition of 3-(fluoromethyl)-phosphoenolpyruvate, preformed 2-oxo-3-butenoate, or 4-fluoro-2-oxobutanoate (which rapidly undergoes elimination of fluoride ion to form 2-oxo-3-butenoate). The inactivating species in all cases is believed to be 2-oxo-3-butenoate. The inactivation is completely prevented by the presence of dithiothreitol, which reacts rapidly with 2-oxo-3-butenoate. Studies with competitive inhibitors of both enzymes indicate that inactivation does not occur at the active site.

1-Carboxyallenyl phosphate, an allenic analogue of phosphoenolpyruvate.

1-Carboxyallenyl phosphate, the allenic homologue of phosphoenolpyruvate, has been synthesized in six steps. The key step in the synthesis is the isomerization of methyl 2-hydroxy-3-butynoate to the corresponding allenol and phosphorylation of this material. The allene is an excellent substrate for pyruvate kinase, undergoing reaction at more than half the rate of phosphoenolpyruvate. The allene is also a substrate for phosphoenolpyruvate carboxylase, being hydrolyzed by the enzyme rather than carboxylated. With both enzymes, the organic product is 2-oxo-3-butenoate, which gradually inactivates the enzymes by reaction with one or more sulfhydryl groups not at the active site.

Aspartic-acid synthesis in C3 plants.

In a previous study (Melzer and O'Leary, 1987, Plant Physiol. 84, 58-60), we used isotopic methods to show that a substantial fraction of protein-bound aspartic acid in tobacco is derived from anaplerotic synthesis via phosphoenolpyruvate (PEP) carboxylase. Similar studies in soybean (Glycine max L.) and spinach (Spinacia oleracea L.) showed a similar pattern, and this pattern persists with age because of slow protein turnover. A more quantitative analysis indicates that about 40% of protein-bound aspartate is derived in this manner. Analyses of free aspartic and malic acids show that contribution of PEP carboxylase to the synthesis of these acids decreases with increasing age. The C4 plant Zea mays L. did not show this pattern.

(E)-3-Cyanophosphoenolpyruvate, a new inhibitor of phosphoenolpyruvate-dependent enzymes.

(E)-3-Cyanophosphoenolpyruvate has been synthesized by reacting dimethyl chlorophosphate with the potassium enolate of ethyl cyanopyruvate. The resulting trialkyl ester was deesterified with bromotrimethylsilane followed by potassium hydroxide. Subsequent treatment with Dowex-50-H+ resin and cyclohexylamine afforded the tricyclohexylammonium salt; only the E geometric isomer was obtained. This compound can be photoisomerized to a 70:30 E:Z mixture. (E)-3-Cyanophosphoenolpyruvate is an excellent competitive inhibitor of phosphoenolpyruvate carboxylase [KI(Mn2+) = 16 microM, KI(Mg2+) = 1360 microM], pyruvate kinase [KI(Mn2+) = 0.085 microM, KI(Mg2+) = 0.76 microM], and enolase [KI(Mn2+) = 360 microM, KI(Mg2+) = 280 microM]. The compound is a substrate for pyruvate kinase (Vmax approximately 1% of phosphoenolpyruvate rate), but not for the other two enzymes. No irreversible inactivation is observed with phosphoenolpyruvate carboxylase of pyruvate kinase.

Anapleurotic CO(2) Fixation by Phosphoenolpyruvate Carboxylase in C(3) Plants.

The role of phosphoenolpyruvate carboxylase in photosynthesis in the C(3) plant Nicotiana tabacum has been probed by measurement of the (13)C content of various materials. Whole leaf and purified ribulose bisphosphate carboxylase are within the range expected for C(3) plants. Aspartic acid purified following acid hydrolysis of this ribulose bisphosphate carboxylase is enriched in (13)C compared to whole protein. Carbons 1-3 of this aspartic acid are in the normal C(3) range, but carbon-4 (obtained by treatment of the aspartic acid with aspartate beta-decarboxylase) has an isotopic composition in the range expected for products of C(4) photosynthesis (-5 per thousand), and it appears that more than half of the aspartic acid is synthesized by phosphoenolpyruvate carboxylase using atmospheric CO(2)/HCO(3) (-). Thus, a primary role of phosphoenolpyruvate carboxylase in C(3) plants appears to be the anapleurotic synthesis of four-carbon acids.

Isotope effect evidence for the zinc hydroxide mechanism of carbonic anhydrase catalysis.

The carbon kinetic isotope effect on the enzymatic dehydration of HCO3- ion is k12/k13 = 1.011 and is independent, within experimental error, of the addition of sucrose, substitution of D2O for H2O, and substitution of enzyme-bound Zn2+ by Co2+. These results are consistent with a ping-pong mechanism in which proton transfer between enzyme and solvent is separated from HCO3- dehydration. For the dehydration half-reaction, diffusional processes are severalfold faster than dehydration, and the rate-determining step is the dehydration itself. The intrinsic isotope effect is approximately 1.011, indicating that hydration of CO2 occurs by reaction of zinc-bound OH-, rather than zinc-bound H2O.

Stereochemistry of reactions catalyzed by glutamate decarboxylase.

When the decarboxylation of L-glutamic acid by the glutamate decarboxylase from Escherichia coli is carried out in D2O, the product gamma-aminobutyric acid contains a single deuterium atom. The stereochemistry of this material was established by conversion to levorotatory methyl 4-phthalimido [4(-2)H] butyrate. The dextrorotatory isomer of the latter compound was synthesized from S-[2(-2)H] glycine by a series of reactions not affecting the stereochemistry at the chiral center. Thus, the decarboxylation of glutamic acid occurs with retention of configuration. Decarboxylation of L-alpha-methylglutamic acid by this enzyme produced levorotatory gamma-aminovaleric acid and thus also occurs with retention of configuration.

Sulfuryl transfer catalyzed by phosphokinases.

Adenosine 5'-sulfatopyrophosphate is a substrate for nucleoside diphosphate kinase. The reaction appears to proceed through a ping-pong mechanism analogous to the physiological reaction involving ATP, presumably by way of a sulfohistidine intermediate. Unlike the phosphoryl transfer reactions, the corresponding sulfuryl transfers catalyzed by nucleoside diphosphate kinase do not have a strict divalent metal requirement. The estimated rate constants for the metal- and nonmetal-catalyzed sulfuryl transfers differ by less than an order of magnitude and are approximately 1000-fold slower than the corresponding phosphate transfers. These results suggest that the role of the metal ion in nucleoside diphosphate kinase is to coordinate the alpha, beta-phosphates of the substrate. Sulfuryl and phosphoryl transfer probably occur through dissociative transition states.

Multiple isotope effect probes of glutamate decarboxylase.

The enzymatic decarboxylation of glutamic acid shows a carbon isotope effect k12/k13 = 1.018 at 37 degree C, pH 4.7. In D2O under otherwise identical conditions, k12/k13 = 1.009. Under the same conditions solvent isotope effects are Vmax H2O/Vmax D2O = 5.0 and (Vmax/Km)H2O/(Vmax/Km)D2O = 2.6. With the assumption that the carbon isotope effect on the decarboxylation step is in the usual range (1.05--1.07), it is possible to derive relative rates and solvent isotope effects for all steps in the enzyme mechanism. Substrate binding in approximately 2-fold weaker in H2O than in D2O, probably because of the desolvation which accompanies binding of the substrate to the enzyme. A proton inventory analysis of the reaction shows that the Schiff base interchange has a large solvent isotope effect composed of relatively small contributions from at least four separate sites. A conformation change probably accompanies this step. The decarboxylation step shows a solvent isotope effect of approximately 2. Schiff base interchange and decarboxylation are both partially rate determining. The pH dependence of the isotope effects indicates that the initial step in the reaction can occur by way of two different pathways.

Medium effects in enzyme-catalyzed decarboxylations.

Carbon isotope effects and steady-state kinetic parameters have been measured for the decarboxylation of arginine and homoarginine by the pyridoxal 5'-phosphate dependent arginine decarboxylase from Escherichia coli. In water at pH 5.25, 5 degrees C, homoarginine shows an isotope effect k12/k13 = 1.601, indicating that the decarboxylation step is entirely rate determining. In the presence of 16 mol % ethylene glycol under otherwise identical conditions, the decarboxylation rate is increased 3-fold, and the carbon isotope effect is 1.044, indicating that the rate of the decarboxylation step is increased by the presence of the less polar solvent. The decarboxylation or arginine under the same conditions shows a similar trend: in water, the isotope effect is 1.027, decreasing to 1.003 in 16% ethylene glycol, with little change in the steady-state rate. Again, the rate of the decarboxylation step is substantially increased by the presence of the nonpolar solvent. Thus, pyridoxal phosphate dependent enzymatic decarboxylations show a medium effect similar to that observed in a number of nonenzymatic decarboxylations. This suggests that these enzymes may accelerate the decarboxylation step by providing a nonpolar environment. Evidence is also presented that desolvation of the substrate carboxyl group may contribute to catalysis.