Stereochemistry of the carboxylation reaction catalyzed by the ATP-dependent phosphoenolpyruvate carboxykinases from Saccharomyces cerevisiae and Anaerobiospirillum succiniciproducens

The stereochemistry of CO(2) addition to phosphoenolpyruvate (PEP) to yield oxaloacetate catalyzed by ATP-dependent Saccharomyces cerevisiae and Anaerobiospirillum succiniciproducens PEP carboxykinases was determined using (Z)-3-fluorophosphoenolpyruvate ((Z)-F-PEP) as a substrate analog. A. succiniciproducens and S. cerevisiae PEP carboxykinases utilized (Z)-F-PEP with 1/14 and 1/47 the respective K(m) values for PEP. On the other hand, in the bacterial and yeast enzymes k(cat) was reduced to 1/67 and 1/48 the value with PEP, respectively. The binding affinity of pyridoxylphosphate-labeled S. cerevisiae and A. succiniciproducens PEP carboxykinases for PEP and (Z)-F-PEP was checked and found to be of similar magnitude for both substrates, suggesting that the lowered K(m) values for the fluorine-containing PEP analog are due to kinetic effects. The lowered k(cat) values when using (Z)-F-PEP as substrate suggest that the electron withdrawing effect of fluorine affects the nucleophilic attack of the double bond of (Z)-F-PEP to CO(2). For the stereochemical analyses, the carboxylation of (Z)-F-PEP was coupled to malate dehydrogenase to yield 3-fluoromalate, which was analyzed by (19)F NMR. The fluoromalate obtained was identified as (2R, 3R)-3-fluoromalate for both the A. succiniciproducens and S. cerevisiae PEP carboxykinases, thus indicating that CO(2) addition to (Z)-F-PEP, and hence PEP, takes place through the 2-si face of the double bond. These results, together with previously published data [Rose, I.A. et al. J. Biol. Chem. 244 (1969) 6130-6133; Hwang, S.H. and Nowak, T. Biochemistry 25 (1986) 5590-5595] indicate that PEP carboxykinases, no matter their nucleotide specificity, catalyze the carboxylation of PEP from the 2-si face of the double bond.     

Mechanistic studies of phosphoenolpyruvate carboxylase from Zea mays utilizing formate as an alternate substrate for bicarbonate.

Formate is an alternate substrate for bicarbonate in the reaction with PEP catalyzed by phosphoenolpyruvate carboxylase from Zea mays, producing formyl phosphate and pyruvate. The Km for formate is 25 +/- 2 mM, and the maximum velocity is 1% of that for bicarbonate at pH 8.0. Use of [18O]formate produces inorganic phosphate containing 1 equiv of 18O, but no label is incorporated into residual phosphoenolpyruvate. PEP carboxylase catalyzes the hydrolysis of phosphoglycolate or L-phospholactate 2000 times more slowly and D-phospholactate 4000 times more slowly than the reaction between bicarbonate and PEP.     

Role of phosphoenolpyruvate carboxylation in Acetobacter xylinum

Glucose-grown cells of Acetobacter xylinum oxidized acetate only when the reaction mixture was supplemented with catalytic quantities of glucose or intermediates of the citrate cycle. Extracts, prepared by sonic treatment, catalyzed the formation of oxalacetate when incubated with phosphoenolpyruvate (PEP) and bicarbonate. Oxalacetate was not formed in the presence of pyruvate plus adenosine triphosphate. The ability to promote carboxylation of PEP was lower in succinate-grown cells than in glucose-grown cells. PEP carboxylase, partially purified from extracts by ammonium sulfate fractionation, catalyzed the stoichiometric formation of oxalacetate and inorganic phosphate from PEP and bicarbonate. The enzyme was not affected by acetyl-coenzyme A or inorganic phosphate. It was inhibited by adenosine diphosphate in a manner competitive with PEP (K(1) = 1.3 mm) and by dicarboxylic acids of the citrate cycle; of these, succinate was the most potent inhibitor. It is suggested that the physiological role of PEP carboxylase in A. xylinum is to affect the net formation of C(4) acids from C(3) precursors, which are essential for the maintainance of the citrate cycle during growth on glucose. The relationship of PEP carboxylase to other enzyme systems metabolizing PEP and oxalacetate in A. xylinum is discussed.     

Phenylphosphate carboxylase: a new C-C lyase involved in anaerobic phenol metabolism in Thauera aromatica

The anaerobic metabolism of phenol in the beta-proteobacterium Thauera aromatica proceeds via carboxylation to 4-hydroxybenzoate and is initiated by the ATP-dependent conversion of phenol to phenylphosphate. The subsequent para carboxylation of phenylphosphate to 4-hydroxybenzoate is catalyzed by phenylphosphate carboxylase, which was purified and studied. This enzyme consists of four proteins with molecular masses of 54, 53, 18, and 10 kDa, whose genes are located adjacent to each other in the phenol gene cluster which codes for phenol-induced proteins. Three of the subunits (54, 53, and 10 kDa) were sufficient to catalyze the exchange of 14CO2 and the carboxyl group of 4-hydroxybenzoate but not phenylphosphate carboxylation. Phenylphosphate carboxylation was restored when the 18-kDa subunit was added. The following reaction model is proposed. The 14CO2 exchange reaction catalyzed by the three subunits of the core enzyme requires the fully reversible release of CO2 from 4-hydroxybenzoate with formation of a tightly enzyme-bound phenolate intermediate. Carboxylation of phenylphosphate requires in addition the 18-kDa subunit, which is thought to form the same enzyme-bound energized phenolate intermediate from phenylphosphate with virtually irreversible release of phosphate. The 54- and 53-kDa subunits show similarity to UbiD of Escherichia coli, which catalyzes the decarboxylation of a 4-hydroxybenzoate derivative in ubiquinone (ubi) biosynthesis. They also show similarity to components of various decarboxylases acting on aromatic carboxylic acids, such as 4-hydroxybenzoate or vanillate, whereas the 10-kDa subunit is unique. The 18-kDa subunit belongs to a hydratase/phosphatase protein family. Phenylphosphate carboxylase is a member of a new family of carboxylases/decarboxylases that act on phenolic compounds, use CO2 as a substrate, do not contain biotin or thiamine diphosphate, require K+ and a divalent metal cation (Mg2+or Mn2+) for activity, and are strongly inhibited by oxygen.     

Synthesis of phosphoenol pyruvate (PEP) analogues and evaluation as inhibitors of PEP-utilizing enzymes.

The synthesis of 10 new phosphoenolpyruvate (PEP) analogues with modifications in the phosphate and the carboxylate function is described. Included are two potential irreversible inhibitors of PEP-utilizing enzymes. One incorporates a reactive chloromethylphosphonate function replacing the phosphate group of PEP. The second contains a chloromethyl group substituting for the carboxylate function of PEP. An improved procedure for the preparation of the known (Z)- and (E)-3-chloro-PEP is also given. The isomers were obtained as a 4 : 1 mixture, resolved by anion-exchange chromatography after the last reaction step. The stereochemistry of the two isomers was unequivocally assigned from the (3)J(H-C) coupling constants between the carboxylate carbons and the vinyl protons. All of these and other known PEP-analogues were tested as reversible and irreversible inhibitors of Mg2+- and Mn2+- activated PEP-utilizing enzymes: enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system (PTS), pyruvate kinase, PEP carboxylase and enolase. Without exception, the most potent inhibitors were those with substitution of a vinyl proton. Modification of the phosphate and the carboxylate groups resulted in less effective compounds. Enzyme I was the least tolerant to such modifications. Among the carboxylate-modified analogues, only those replaced by a negatively charged group inhibited pyruvate kinase and enolase. Remarkably, the activity of PEP carboxylase was stimulated by derivatives with neutral groups at this position in the presence of Mg2+, but not with Mn2+. For the irreversible inhibition of these enzymes, (Z)-3-Cl-PEP was found to be a very fast-acting and efficient suicide inhibitor of enzyme I (t(1/2) = 0.7 min).     

ATPase activity of biotin carboxylase provides evidence for initial activation of HCO3- by ATP in the carboxylation of biotin

When we incubated biotin carboxylase from Escherichia coli with ATP in absence of biotin we observed HCO3- -dependent ATP hydrolysis, which was activated by 10% ethanol in the same proportion as the activity of D-biotin carboxylation assayed in the presence of biotin. The two activities exhibited identical heat stability and were protected equally by glycerol; both required Mg2+ and K+ and showed similar dependency on the concentration of ATP. Biotin assay excluded potential contamination by traces of biotin as a cause of the observed ATP hydrolysis, and this was confirmed by the findings that carboxybiotin did not accumulate and that avidin was uninhibitory. Therefore we concluded that this HCO3- -dependent ATPase was genuinely a partial activity of biotin carboxylase. This partial activity supports a sequential mechanism for enzymatic carboxylation of biotin in which HCO3- is activated by ATP in a first step. It is consistent with the initial formation of the carbonic-phosphoric anhydride (HOCO2PO3(2-)), and it does not agree with models where biotin is phosphorylated by ATP prior to reaction with HCO3-. It appears that enzymes that use HCO3- for carboxylation, including biotin-dependent carboxylases, phosphoenolpyruvate carboxylase, and carbamoyl phosphate synthetase, activate HCO3- by a common mechanism involving the initial formation of the carbonic-phosphoric anhydride.     

Fractionation of stable carbon isotopes by phosphoenolpyruvate carboxylase from c(4) plants

The active species of "CO(2)" and the amount of fractionation of stable carbon isotopes have been determined for a partially purified preparation of phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) from corn (Zea mays) leaves. The rates of the enzyme reactions, using substrate amounts of HCO(3) (-), CO(2) or CO(2) plus carbonic anhydrase, show that HCO(3) (-) is the active species of "CO(2)" utilized by PEP carboxylase. The K(m) values for CO(2) and HCO(3) (-) are 1.25 mm and 0.11 mm, respectively, which further suggest the preferential utilization of HCO(3) (-) by PEP carboxylase. The amount of fractionation of stable carbon isotopes by PEP carboxylase from an infinite pool of H(12)CO(3) (-) and H(13)CO(3) (-) was -2.03 per thousand. This enzyme fractionation (delta), together with the fractionation associated with absorption of CO(2) into plant cells and the equilibrium fractionation associated with atmospheric CO(2) and dissolved HCO(3) (-) are discussed in relation to the fractionation of stable carbon isotopes of atmospheric CO(2) during photosynthesis in C(4) plants.     

Synthesis and study of (Z)-3-chlorophosphoenolpyruvate

(Z)-3-Chlorophosphoenolpyruvate has been synthesized by the reaction of 3,3-dichloropyruvic acid with trimethylphosphite, followed by deesterification. This compound is a competitive inhibitor of pyruvate kinase and phosphoenolpyruvate carboxylase. Pyruvate kinase is not inactivated upon prolonged incubation with the compound, but phosphoenolpyruvate carboxylase is slowly inactivated (t1/2 = 5 h). The compound is a substrate for both enzymes, being acted upon by pyruvate kinase approximately 0.1% as rapidly as phosphoenolpyruvate itself. In the case of phosphoenolpyruvate carboxylase, the compound is converted into a 3:1 mixture of chloropyruvate and chlorooxalacetate, at an overall rate that is about 25% the carboxylation rate for phosphoenolpyruvate.     

Carbon-13 and deuterium isotope effects on the catalytic reactions of biotin carboxylase

13C and 2H kinetic isotope effects have been used to investigate the mechanism of enzymic biotin carboxylation. D(V/K) is 0.50 in 80% D2O at pD 8.0 for the forward reaction and 0.57 at pD 8.5 for the phosphorylation of ADP by carbamoyl phosphate. These values approach the theoretical maximum limit for a reaction in which a proton is transferred from a sulfhydryl to a nitrogen or oxygen base. Therefore, it appears that this portion of the reaction is at or near equilibrium. 13(V/K) at pH 8 is 1.007; the small magnitude of this number suggests that the reaction is almost fully committed by the time the carbon-sensitive steps are reached. There does not appear to be a reverse commitment to the reaction under the conditions in which 13(V/K) was determined. A large forward commitment is consistent with the failure to observe positional isotope exchange from the beta gamma-bridge position to the beta-nonbridge position in [18O4]ATP or washout of 18O from the gamma-nonbridge positions. Transfer of 18O from bicarbonate to inorganic phosphate in the forward reaction was clearly observed, however. These observations suggest that biotin carboxylase exists in two distinct forms which differ in the protonation states of the two active-site bases, one of which is a sulfhydryl. Only when the sulfhydryl is ionized and the second base protonated can catalysis take place. Carboxylation of biotin is postulated to occur via a pathway in which carboxyphosphate in formed by nucleophilic attack of bicarbonate on ATP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Vitamin K-dependent carboxylation. A synthetic peptide based upon the gamma-carboxylation recognition site sequence of the prothrombin propeptide is an active substrate for the carboxylase in vitro

The vitamin K-dependent blood-clotting proteins contain a gamma-carboxylation recognition site in the propeptide, between the signal peptide and the mature protein, that directs gamma-carboxylation of specific glutamic acid residues. To develop a better substrate for the in vitro assay of the vitamin K-dependent gamma-carboxylase and to understand the substrate recognition requirements of the carboxylase, we prepared synthetic peptides based upon the structure of human proprothrombin. These peptides were employed as substrates for in vitro carboxylation using a partially purified form of the bovine liver carboxylase. A 28-residue peptide (HVFLAPQQARSLLQRVRRANTFLEEVRK), based on residues -18 to +10 in proprothrombin, includes the complete propeptide and the first 10 residues of acarboxyprothrombin. Carboxylation of this peptide is characterized by a Km of 3.6 microM. In contrast, FLEEL is carboxylated with a Km of about 2200 microM. A 10-residue peptide (ANTFLEEVRK), based on residues +1 to +10 in prothrombin, and a 20-residue peptide (ARSLLQRVRRANTFLEEVRK), based on residues -10 to +10 in proprothrombin, are also poor substrates for the carboxylase. Replacement of phenylalanine with alanine at residue 3 (equivalent to position -16 in proprothrombin) in the 28-residue peptide significantly alters the Km to 200 microM. A synthetic propeptide (HVFLAPQQARSLLQRVRRY), homologous to residues -18 to -1 in proprothrombin, inhibited carboxylation of the 28-residue peptide substrate with a Ki of 3.5 microM, but modestly stimulated the carboxylation of the 5- and 10-residue peptide substrates. These results indicate that an intact carboxylation recognition site is required for efficient in vitro carboxylation and that this site includes critical residues in region -18 to -11 of proprothrombin. The carboxylation recognition site in the propeptide binds directly to the carboxylase or to a closely associated protein.     

Vitamin K-dependent carboxylation. In vitro modification of synthetic peptides containing the gamma-carboxylation recognition site

Synthetic peptides including the gamma-carboxylation recognition site and acidic amino acids were compared as substrates for vitamin K-dependent gamma-carboxylation by bovine liver carboxylase. The 28-residue proPT28 (proprothrombin -18 to +10) and proFIX28 (pro-Factor IX -18 to +10) were carboxylated with a Km of 3 microM. The Vmax of proPT28 was 2-3 times greater than that of proFIX28. An analog of proFIX28 that contained the prothrombin propeptide had a Vmax 2-3-fold greater than an analog of proPT28 that contained the Factor IX propeptide. proFIX28/RS-1, based upon Factor IX Cambridge, proFIX28/RQ-4, based upon Factor IX Oxford 3, and proFIX28 had equivalent Km and Vmax values. Analogs of proPT28 containing Ala6-Glu7 or Glu6-Ala7 were carboxylated at equivalent rates. A peptide containing Asp6-Asp7 was carboxylated at a rate of about 1% of that of Glu carboxylation. Carboxylation of peptides containing Asp6-Glu7 and Glu6-Asp7 yielded results identical with peptides containing Ala6-Glu7 and Glu6-Ala7. Carboxymethylcysteine was not carboxylated when substituted for Glu6 in a peptide containing Asp7. These results indicate that the prothrombin propeptide is more efficient in the carboxylation process than is the Factor IX propeptide, but that both propeptides direct carboxylation; the gamma-carboxylation recognition site does not include residues -4 and -1; aspartic acid and carboxymethylcysteine are poor substrates for the carboxylase, but aspartic acid does not inhibit the carboxylation of adjacent glutamic acids.     

Carboxylation and dephosphorylation of phosphoenol-3-fluoropyruvate by maize leaf phosphoenolpyruvate carboxylase.

The analogue (Z)-phosphoenol-3-fluoropyruvate [(Z)-3-fluoro-2-(phosphono-oxy)propenoic acid] was tested as substrate of maize leaf phosphoenolpyruvate carboxylase. Studies with NaH14CO3 indicate that the analogue is carboxylated by the enzyme. However, this reaction accounts for only one-tenth of the activity measured by Pi liberation. The rest of the analogue is merely dephosphorylated. This is the first analogue for which both carboxylation and dephosphorylation have been observed.     

Interaction of acetyl phosphate and carbamyl phosphate with plant phosphoenolpyruvate carboxylase.

Acetyl phosphate produced an increase in the maximum velocity (Vmax. for the carboxylation of phosphoenolpyruvate catalysed by phosphoenolpyruvate carboxylase. The limiting Vmax. was 22.2 mumol X min-1 X mg-1 (185% of the value without acetyl phosphate). This compound also decreased the Km for phosphoenolpyruvate to 0.18 mM. The apparent activation constants for acetyl phosphate were 1.6 mM and 0.62 mM in the presence of 0.5 and 4 mM-phosphoenolpyruvate respectively. Carbamyl phosphate produced an increase in Vmax. and Km for phosphoenolpyruvate. The variation of Vmax./Km with carbamyl phosphate concentration could be described by a model in which this compound interacts with the carboxylase at two different types of sites: an allosteric activator site(s) and the substrate-binding site(s). Carbamyl phosphate was hydrolysed by the action of phosphoenolpyruvate carboxylase. The hydrolysis produced Pi and NH4+ in a 1:1 relationship. Values of Vmax. and Km were 0.11 +/- 0.01 mumol of Pi X min-1 X mg-1 and 1.4 +/- 0.1 mM, respectively, in the presence of 10 mM-NaHCO3. If HCO3- was not added, these values were 0.075 +/- 0.014 mumol of Pi X min-1 X mg-1 and 0.76 +/- 0.06 mM. Vmax./Km showed no variation between pH 6.5 and 8.5. The reaction required Mg2+; the activation constants were 0.77 and 0.31 mM at pH 6.5 and 8.5 respectively. Presumably, carbamyl phosphate is hydrolysed by phosphoenolpyruvate carboxylase by a reaction the mechanism of which is related to that of the carboxylation of phosphoenolpyruvate.     

Effects of the Phosphoenolpyruvate Carboxylase Inhibitor 3,3-Dichloro-2-(Dihydroxyphosphinoylmethyl)propenoate on Photosynthesis: C(4) Selectivity and Studies on C(4) Photosynthesis

The effect of 3,3-dichloro-2-(dihydroxyphosphinoylmethyl)-propenoate (DCDP), an analog of phosphoenolpyruvate (PEP), on PEP carboxylase activity in crude leaf extracts and on photosynthesis of excised leaves was examined. DCDP is an effective inhibitor of PEP carboxylase from Zea mays or Panicum miliaceum; 50% inhibition was obtained at 70 or 350 micromolar, respectively, in the presence of 1 millimolar PEP and 1 millimolar HCO₃⁻. When fed to leaf sections via the transpiration stream, DCDP at 1 millimolar strongly inhibited photosynthesis in C₄ species (79-98% inhibition for a range of seven C₄ species), but only moderately in C₃ species (12-46% for four C₃ species), suggesting different mechanisms of inhibition for each photosynthetic type. The response of P. miliaceum (C₄) net photosynthesis to intercellular pCO₂ showed that carboxylation efficiency, as well as the CO₂ saturated rate, are lowered in the presence of DCDP and supported the view that carboxylation efficiency in C₄ species is directly related to PEP carboxylase activity. A fivefold increase in intercellular pCO₂ over that occurring in P. miliaceum under normal photosynthesis conditions only increased net photosynthesis rate in the presence of 1 millimolar DCDP from zero to about 5% of the maximal uninhibited rate. Therefore, it seems unlikely that direct fixation of atmospheric CO₂ by the bundle sheath cells makes any significant contribution to photosynthetic CO₂ assimilation in C₄ species. The results support the concept that C₄-selective herbicides may be developed based on inhibitors of C₄ pathway reactions.     

Binding of the factor IX gamma-carboxyglutamic acid domain to the vitamin K-dependent gamma-glutamyl carboxylase active site induces an allosteric effect that may ensure processive carboxylation and regulate the release of carboxylated product

Propeptides of the vitamin K-dependent proteins bind to an exosite on gamma-glutamyl carboxylase; while they are bound, multiple glutamic acids in the gamma-carboxyglutamic acid (Gla) domain are carboxylated. The role of the propeptides has been studied extensively; however, the role of the Gla domain in substrate binding is less well understood. We used kinetic and fluorescence techniques to investigate the interactions of the carboxylase with a substrate containing the propeptide and Gla domain of factor IX (FIXproGla41). In addition, we characterized the effect of the Gla domain and carboxylation on propeptide and substrate binding. For the propeptide of factor IX (proFIX18), FIXproGla41, and carboxylated FIXproGla41, the Kd values were 50, 2.5, and 19.7 nM and the koff values were 273 x 10(-5), 9 x 10(-5), and 37 x 10(-5) s(-1), respectively. The koff of proFIX18 is reduced 3-fold by FLEEL and 9-fold by the Gla domain (residues 1-46) of FIX. The pre-steady state rate constants for carboxylation of FIXproGla41 was 0.02 s(-1) in enzyme excess and 0.016 s(-1) in substrate excess. The steady state rate in substrate excess is 4.5 x 10(-4) s(-1). These results demonstrate the following. 1) The pre-steady state carboxylation rate constant of FIXproGla41 is significantly slower than that of FLEEL. 2) The Gla domain plays an allosteric role in substrate-enzyme interactions. 3) Carboxylation reduces the allosteric effect. 4) The similarity between the steady state carboxylation rate constant and product dissociation rate constant suggests that product release is rate-limiting. 5) The increased dissociation rate after carboxylation contributes to the release of product.     

Regulation of pyruvate metabolism via pyruvate carboxylase in rat brain mitochondria

1. The fixation of CO(2) by pyruvate carboxylase in isolated rat brain mitochondria was investigated. 2. In the presence of pyruvate, ATP, inorganic phosphate and magnesium, rat brain mitochondria fixed H(14)CO(3) (-) into tricarboxylic acid-cycle intermediates at a rate of about 250nmol/30min per mg of protein. 3. Citrate and malate were the main radioactive products with citrate containing most of the radioactivity fixed. The observed rates of H(14)CO(3) (-) fixation and citrate formation correlated with the measured activities of pyruvate carboxylase and citrate synthase in the mitochondria. 4. The carboxylation of pyruvate by the mitochondria had an apparent K(m) for pyruvate of about 0.5mm. 5. Pyruvate carboxylation was inhibited by ADP and dinitrophenol. 6. Malate, succinate, fumarate and oxaloacetate inhibited the carboxylation of pyruvate whereas glutamate stimulated it. 7. The results suggest that the metabolism of pyruvate via pyruvate carboxylase in brain mitochondria is regulated, in part, by the intramitochondrial concentrations of pyruvate, oxaloacetate and the ATP:ADP ratio.     

Novel insights into the biotin carboxylase domain reactions of pyruvate carboxylase from Rhizobium etli

The catalytic mechanism of the MgATP-dependent carboxylation of biotin in the biotin carboxylase domain of pyruvate carboxylase from R. etli (RePC) is common to the biotin-dependent carboxylases. The current site-directed mutagenesis study has clarified the catalytic functions of several residues proposed to be pivotal in MgATP-binding and cleavage (Glu218 and Lys245), HCO(3)(-) deprotonation (Glu305 and Arg301), and biotin enolization (Arg353). The E218A mutant was inactive for any reaction involving the BC domain and the E218Q mutant exhibited a 75-fold decrease in k(cat) for both pyruvate carboxylation and the full reverse reaction. The E305A mutant also showed a 75- and 80-fold decrease in k(cat) for both pyruvate carboxylation and the full reverse reaction, respectively. While Glu305 appears to be the active site base which deprotonates HCO(3)(-), Lys245, Glu218, and Arg301 are proposed to contribute to catalysis through substrate binding interactions. The reactions of the biotin carboxylase and carboxyl transferase domains were uncoupled in the R353M-catalyzed reactions, indicating that Arg353 may not only facilitate the formation of the biotin enolate but also assist in coordinating catalysis between the two spatially distinct active sites. The 2.5- and 4-fold increase in k(cat) for the full reverse reaction with the R353K and R353M mutants, respectively, suggests that mutation of Arg353 allows carboxybiotin increased access to the biotin carboxylase domain active site. The proposed chemical mechanism is initiated by the deprotonation of HCO(3)(-) by Glu305 and concurrent nucleophilic attack on the γ-phosphate of MgATP. The trianionic carboxyphosphate intermediate formed reversibly decomposes in the active site to CO(2) and PO(4)(3-). PO(4)(3-) then acts as the base to deprotonate the tethered biotin at the N(1)-position. Stabilized by interactions between the ureido oxygen and Arg353, the biotin-enolate reacts with CO(2) to give carboxybiotin. The formation of a distinct salt bridge between Arg353 and Glu248 is proposed to aid in partially precluding carboxybiotin from reentering the biotin carboxylase active site, thus preventing its premature decarboxylation prior to the binding of a carboxyl acceptor in the carboxyl transferase domain.     

Stereochemistry of phosphoenolpyruvate carboxylation catalyzed by phosphoenolpyruvate carboxykinase

The stereochemistry of the carboxylation of phosphoenolpyruvate to yield oxalacetate, catalyzed by chicken liver phosphoenolpyruvate carboxykinase and by Ascaris muscle phosphoenolpyruvate carboxykinase, was determined. The substrate (Z)-3-fluorophosphoenolpyruvate was used for the stereochemical analysis. The carboxylation reaction was coupled to malate dehydrogenase to yield 3-fluoromalate, and the stereochemistry of the products was identified by 19F NMR. In separate experiments, the enantiomeric tautomers of 3-fluorooxalacetate were shown to be utilized by malate dehydrogenase to yield (2R,3R)- and (2R,3S)-3-fluoromalate in nearly identical amounts. The products were identified by 19F NMR. When (Z)-3-fluorophosphoenolpyruvate was used as a substrate for phosphoenolpyruvate carboxykinase from avian liver and from Ascaris, and malate dehydrogenase was used to trap the product, only a single diastereomer was observed. This product was shown to be (2R,3R)-3-fluoromalate in each case. The assignments were based on coupling constants taken from Keck et al. [Keck, R., Hess, H., & Rétey, J. (1980) FEBS Lett. 114, 287]. These results indicate that the stereochemistry of carboxylation, catalyzed by chicken phosphoenolpyruvate carboxykinase and by Ascaris phosphoenolpyruvate carboxykinase, is identical and takes place from the si side of the enzyme-bound phosphoenolpyruvate. The carboxylation reaction was run both in H2O and in D2O. No deuterium incorporation into fluoromalate was shown to occur. The product 3-fluorooxalacetate is thus released from phosphoenolpyruvate carboxykinase as the keto form and is reduced more rapidly by reduced nicotinamide adenine dinucleotide with malate dehydrogenase than by the occurrence of tautomerization.     

A kinetic investigation of phosphoenolpyruvate carboxylase from Zea mays.

The reaction catalyzed by phosphoenolpyruvate carboxylase from Zea mays has been studied kinetically. Results of initial velocity patterns and inhibition studies indicate that phosphoenolpyruvate carboxylase has a random sequential mechanism in which there is a high level of synergism in the binding of substrates. The preferred order of addition of reactants is Mg2+, phosphoenolpyruvate, and bicarbonate. The binding of Mg2+ is at equilibrium. Values for the various kinetic parameters are KiMg = 2.3 +/- 0.4 mM, KPEP = 3.6 +/- 0.6 mM, KiPEP = 0.2 +/- 0.07 mM, and Kbicarbonate = 0.18 +/- 0.04 mM. In addition, double inhibition experiments have been performed to examine the nature of the active site interactions with the putative intermediates, carboxy phosphate and the enolate of pyruvate. Highly synergistic inhibition of phosphoenolpyruvate carboxylase was observed in the presence of oxalate and carbamyl phosphate (alpha = 0.0013). However, an antisynergistic relationship exists between oxalate and phosphonoformate (alpha = 2.75).     

The use of (E)- and (Z)-phosphoenol-3-fluoropyruvate as mechanistic probes reveals significant differences between the active sites of KDO8P and DAHP synthases

The enzymes 3-deoxy-d-manno-2-octulosonate-8-phosphate (KDO8P) synthase and 3-deoxy-d-arabino-2-heptulosonate-7-phosphate (DAHP) synthase catalyze a similar aldol-type condensation between phosphoenolpyruvate (PEP) and the corresponding aldose: arabinose 5-phosphate (A5P) and erythrose 4-phosphate (E4P), respectively. While KDO8P synthase is metal-dependent in one class of organisms and metal-independent in another, only a metal-dependent class of DAHP synthases has thus far been identified in nature. We have used catalytically active E and Z isomers of phosphoenol-3-fluoropyruvate [(E)- and (Z)-FPEP, respectively] as mechanistic probes to characterize the differences and/or the similarities between the metal-dependent and metal-independent KDO8P synthases as well as between the metal-dependent KDO8P synthase and DAHP synthase. The direct evidence of the overall stereochemistry of the metal-dependent Aquifex pyrophilus KDO8P synthase (ApKDO8PS) reaction was obtained by using (E)- and (Z)-FPEPs as alternative substrates and by subsequent (19)F NMR analysis of the products. The results reveal the si face addition of the PEP to the re face of the carbonyl of A5P, and establish that the stereochemistry of ApKDO8PS is identical to that of the metal-independent Escherichia coli KDO8P synthase enzyme (EcKDO8PS). In addition, both ApKDO8PS and EcKDO8PS enzymes exhibit high selectivity for (E)-FPEP versus (Z)-FPEP, the relative k(cat)/K(m) ratios being 100 and 33, respectively. In contrast, DAHP synthase does not discriminate between (E)- and (Z)-FPEP (the k(cat)/K(m) being approximately 7 x 10(-)(3) microM(-)(1) s(-)(1) for both compounds). The pre-steady-state burst experiments for EcKDO8PS showed that product release is rate-limiting for the reactions performed with either PEP, (E)-FPEP, or (Z)-FPEP, although the rate constants, for both product formation and product release, were lower for the fluorinated analogues than for PEP [125 and 2.3 s(-)(1) for PEP, 2.5 and 0.2 s(-)(1) for (E)-FPEP, and 9 and 0.1 s(-)(1) for (Z)-FPEP, respectively]. The observed data indicate substantial differences in the PEP subsites and open the opportunity for the design of selective inhibitors against these two families of enzymes.