Rate Of Product Release Research Articles

Mechanism of action of Escherichia coli formamidopyrimidine N-glycosylase: Role of K155 in substrate binding and product release

Escherichia coli formamidopyrimidine N-glycosylase (fpg) is a DNA glycosylase with an associated beta,delta-lyase activity. We have recently shown that the highly conserved lysine residue K155 is important for base recognition. Incubation of a double-stranded DNA containing an abasic site with the wild-type fpg protein generated only beta,delta-product. However, incubation of a double-stranded DNA containing an abasic site opposite a small gap with fpg protein generated predominantly beta-product. These data suggested that the induction of a double-strand break by fpg led to the destabilization of the protein-DNA covalent intermediate, causing the fpg protein to prematurely dissociate from the DNA substrate. Furthermore, when a double-stranded DNA containing an abasic site opposite an A was used as a substrate, K155A mutant fpg protein yielded a mixture of beta- and beta,delta-products. These data suggested that K155 is essential for maintaining the stability of the intermediary protein-DNA covalent complex. Pre-steady-state burst kinetics showed that mutation in K155 led to the apparent disappearance of the initial burst, suggesting that the rate of product release from K155A is much greater than the rate of chemical reaction catalyzed by the mutant enzyme. This is consistent with the idea that K155A dissociates prematurely from the covalent complex, leading to a higher turnover number observed for K155A for DNA substrate containing an AP site.

Pre-steady-state kinetics of initiation of transcription by T7 RNA polymerase: a new kinetic model

In order to begin to understand the mechanism of the initiation of transcription in the model bacteriophage T7 RNA polymerase system, the simplest possible reaction, the synthesis of a dinucleotide, has been followed by quench-flow kinetics and numerical integration of mechanism-specific rate equations has been used to test specific kinetic models. In order to fit the observed time dependence in the pre-steady-state kinetics, a model for dinucleotide synthesis is proposed in which rebinding of the dinucleotide to the enzyme-DNA complex must be included. Separate reactions using dinucleotide as a substrate confirm this mechanism and the determined rate constants. The dinucleotide rebinding observed as inhibition under these conditions forms a productive intermediate in the synthesis of longer transcripts, and must be included in future kinetic mechanisms. The rate-limiting step leading to product formation shows a substrate dependence consistent with the binding of two substrate GTP molecules, and at saturating levels of GTP, is comparable in magnitude to the product release rate. The rate of product release shows a positive correlation with the concentration of GTP, suggesting that the reaction shows base-specific substrate activation. The binding of another substrate molecule, presumably via interaction with the triphosphate binding site, likely facilitates displacement of the dinucleotide product from the complex.

Activation of the insulin receptor's kinase domain changes the rate-determining step of substrate phosphorylation.

The insulin receptor and many other protein kinases are activated by relief of intrasteric inhibition that is regulated by reversible phosphorylation. The changes accompanying activation of the insulin receptor's kinase domain were analyzed using steady-state kinetics, viscometric analysis, and equilibrium binding measurements. Peptide phosphorylation catalyzed by the unphosphorylated basal-state kinase is limited by a slow rate of the chemical step, and the activated enzyme is limited by product release rates. Underlying these changes were a 36-fold increase in the rate constant for the chemical step of the enzyme-catalyzed reaction, a 5-fold increase in the affinity for MgATP, and an 8-fold increase in the affinity for peptide substrate. This results in binding of substrates that is 2.2 kcal/mol more favorable and a free energy barrier for transition state formation that is lowered by 2.1 kcal/mol in the activated enzyme. Therefore, the change in conformational free energy inherent in the protein after autophosphorylation [Bishop, S. M., Ross, J. B. A., and Kohanski, R. A. (1999) Biochemistry 38, 3079-3089] is equally distributed between formation of the substrate ternary complex and formation of the transition state complex.

Refolding of rRNA exons enhances dissociation of the Tetrahymena intron.

Self-splicing of the Tetrahymena pre-rRNA is inhibited by a conserved rRNA hairpin P(-1) upstream of the 5' splice site. P(-1) inhibits self-splicing by competing with formation of the P1 splice site helix. Here we show that the P(-1) hairpin also enhances dissociation of the spliced products, which was monitored by native gel electrophoresis. Mutations that stabilize the rRNA hairpin increase the rate of dissociation approximately 10-fold, from 0.5 min(-1) for the wild-type RNA to approximately 4 min(-1) at 30 degrees C. Conversely, mutations or oligonucleotides that inhibit refolding of the exons and that stabilize the P1 helix decrease the rate of product release. The results suggest that refolding of products can be used to stimulate the turnover of ribozyme-catalyzed reactions. In the pre-rRNA, this conformational change helps shift the equilibrium of self-splicing toward the mature rRNA.

Conformational Cycle of a Single Working Enzyme

By simultaneous analysis of the on-time distribution and autocorrelation function of a single working cholesterol oxidase enzyme, a diffusional model reveals the coupling of conformation change with enzyme action. Active-site oxidation induces a conformational change that opens the path for substrate entry. Its binding, in turn, induces the reverse protein relaxation process, which tightens the active site, thereby reducing the rate of product release.

A MELCOR-Based Model to Calculate Vapor Natural Circulation in Pressurized Water Reactor High-Pressure Severe Accidents

The ability of the probabilistic safety assessment code MELCOR 1.8.2 to deal with station blackout accidents, characterized by prolonged in-vessel and primary system vapor natural circulation, is analyzed. Results of the analysis recommended a modification of the gravitational term in the momentum equation and the inclusion of the convective term to capture in-vessel natural circulation. Moreover, certain guidelines to build the thermal-hydraulic and core degradation numerical meshes must be respected. A model is proposed that has been applied to simulate the Three Mile Island Unit 2 phase 2 accident, for which natural circulation flows were supposed to take place. The compatibility of the establishment of natural circulation flow with accident measurements and estimations is observed. Furthermore, core degradation results seem reasonable at first sight, although improvements concerning these models are suggested.The ability of the model to cope with a full sequence in a commercial plant is demonstrated: A station blackout for a one-loop pressurized water reactor was calculated from the initial event to the instant of primary system failure. In-vessel and ex-vessel natural circulation flows of vapor are automatically established, and heatup and fission product release rates are estimated.

Stopped-flow kinetic analysis of the ligand-induced coil-helix transition in glutathione S-transferase A1-1: evidence for a persistent denatured state.

Structural studies have suggested that the glutathione S-transferase (GST) A1-1 isozyme contains a dynamic C-terminus which undergoes a ligand-dependent disorder-order transition and sequesters substrates within the active site. Here, the contribution of the C-terminus to the kinetics and thermodynamics of ligand binding and dissociation has been determined. Steady-state turnover rates of the wild type (WT) and a C-terminal truncated (Delta209-222) rGST A1-1 with ethacrynic acid (EA) were measured in the presence of variable concentrations of viscogen. The results indicate that a physical step involving segmental protein motion is at least partially rate limiting at temperatures between 10 and 40 degrees C for WT. Dissociation rates of the glutathione-ethacrynic acid product conjugate (GS-EA), determined by stopped-flow fluorescence, correspond to the steady-state turnover rates. In contrast, the chemical step governs the turnover reaction by Delta209-222, suggesting that the slow rate of product release for WT is controlled by the dynamics of the C-terminal coil-helix transition. In addition, the association reaction of WT rGST A1-1 with GS-EA established that the binding was biphasic and included ligand docking followed by slow isomerization of the enzyme-ligand complex. In contrast, binding of GS-EA to Delta209-222 was a monophasic, bimolecular reaction. These results indicate that the binding of GS-EA to WT rGST A1-1 proceeds via an induced fit mechanism, with a slow conformational step that corresponds to the coil-helix transition. However, the biphasic dissociation kinetics for the wild type, and the recovered kinetic parameters, suggest that a significant fraction of the [GST.GS-EA] complex ( approximately 15%) retains a persistent disordered state at equilibrium.

Pre-steady-state and steady-state kinetic studies on transcription initiation catalyzed by T7 RNA polymerase and its active-site mutants K631R and Y639F.

The kinetic mechanism of transcription initiation was studied under conditions that allow a single nucleotide addition to an initiating dinucleotide without interference of enzyme-DNA dissociation or protein recycling. Pre-steady-state kinetic studies have provided polymerization rate constants of 3.9, 5.9, and 3.9 s-1, reverse polymerization rate constants of 3.2, 2.1, and 2.8 s-1, and dissociation constants for the incoming nucleotide of 26, 49, and 24 microM at 21 degreesC, respectively, for the wild type and its active-site mutants K631R and Y639F. The results suggest a model in which K631 interacts with the phosphate group(s) of the incoming substrate. The internal equilibrium constants for the bound species are close to unity, consistent with the values for other phosphoryl transfer enzymes. The rate constants for chemical bond formation are at least 50 times higher than the rate constants for product dissociation. The product release rate constants, k3, are comparable to the steady-state rates, suggesting that the rate-determining step for all three enzymes may be a product dissociation step. The existence of two possible conformers E and E' that are in rapid equilibrium is postulated, to reconcile reduced burst sizes with full activity of the mutant enzymes. Both forms can form the quaternary complex, but only the E form is capable of catalyzing phosphodiester bond formation. The fraction of the catalytically active E form varies from essentially 100% for the wild type to 38 and 32% for the mutants K631R and Y639F, respectively. Upon entering the elongation phase, the E form becomes the dominant form in all three enzymes, leading to comparable rates of elongation for the wild type and Y639F mutant. The rate of synthesis of long transcripts is markedly diminished for the K631R mutant due to decreased processivity.

A second magnesium ion is critical for ATP binding in the kinase domain of the oncoprotein v-Fps.

The activity of the kinase domain of the oncoprotein v-Fps was found to be sensitive to the concentration of magnesium ions. Plots of initial velocity versus free magnesium concentration are hyperbolic and do not extrapolate to the origin at stoichiometric ATP-Mg, indicating that there are two sites for metal chelation on the enzyme and the second is nonessential for catalysis. The second metal is strongly activating and increases the reaction rate constant almost 20-fold from 0.5 to 8.3 s-1 using 0.2 mM ATP-Mg and 1 mM peptide, EAEIYEAIE. This increase in rate is due to a large increase in the apparent affinity of ATP-Mg at high magnesium concentrations. At 0.5 and 10 mM free Mg2+, KATP-Mg is 3.6 and 0.22 mM, respectively. Extrapolation of the observed affinity of ATP-Mg to zero and infinite free metal indicates that KATP-Mg is greater than 8 mM in the absence of the second metal and 0.1 mM in the presence of the second metal, a minimum 80-fold enhancement. By comparison, free levels of the divalent ion do not influence maximum turnover (kcat) and have only a 2-fold effect on the Km for the peptide substrate between 0.5 and 20 mM free Mg2+. Viscosometric studies indicate that free Mg2+ does not influence the rates of phosphoryl transfer or net product release above 0.5 mM but does affect directly the dissociation constant for ATP-Mg. The Kd for ATP-Mg in the absence and presence of the second metal ion is >32 and 0.4 mM, respectively. At high magnesium concentrations, ATP-Mg and the peptide substrate bind independently, while at lower concentrations (0.5 mM), there is significant negative binding synergism suggesting that the second metal may help to reduce charge repulsion between ATP-Mg and the peptide. The data indicate that the first metal is sufficient for phosphoryl transfer. While the second metal could have some influence on phosphoryl transfer or product binding, it is a potent activator that functions minimally by controlling ATP-Mg binding.

Flow rate-modulated dissolution of fibrin with clot-embedded and circulating proteases.

The efficiency of plasmin, miniplasmin, and neutrophil leukocyte elastase in fibrin digestion is well characterized in static systems. Since in vivo the components of the fibrinolytic system are permanently exposed to flow, we have developed two in vitro models and studied the effect of shear forces on fibrin dissolution with these proteases. Cylindrical nonocclusive fibrin clots are perfused at various flow rates through their preformed axial channel, and dissolution of fibrin is followed by measuring the absorbance of degradation products released into the circulating fluid phase. In one experimental setting, fibrin surface is degraded with enzymes applied in the recirculating fluid phase; in another setting, clots containing gel-embedded proteases are perfused with enzyme-free buffer. As shear rate at fibrin surface is changed from 25 to 500 s(-1), the rate of product release by recirculated enzymes increases 2.8-, 2.9-, and 4-fold for plasmin, miniplasmin, and porcine pancreatic elastase, respectively. Buffer-perfused fibrin containing gel-embedded plasmin or miniplasmin is disintegrated by shear forces at a relatively early stage of dissolution, and this disassembly is related to the formation of fragment Y (150 kDa) and fragment D (100 kDa) fibrin degradation products. Fibrin clots degraded by incorporated polymorphonuclear leukocyte elastase, which yields different degradation products, do not disassemble abruptly, even at the highest shear rate (500 s(-1)). Our results suggest that fibrin surface degradation is accelerated with increasing shear rate and that plasmin or miniplasmin embedded in the clot promotes the release of particular clot remnants into the circulating phase, whereas polymorphonuclear leukocyte elastase does not.

ATP Analogs and Muscle Contraction: Mechanics and Kinetics of Nucleoside Triphosphate Binding and Hydrolysis

The mechanical behavior of skinned rabbit psoas muscle fiber contractions and in vitro motility of F-actin ( V f) have been examined using ATP, CTP, UTP, or their 2-deoxy forms (collectively designated as nucleotide triphosphates or NTPs) as contractile substrates. Measurements of actin-activated heavy meromyosin (HMM) NTPase, the rates of NTP binding to myosin and actomyosin, NTP-mediated acto-HMM dissociation, and NTP hydrolysis by acto-HMM were made for comparison to the mechanical results. The data suggest a very similar mechanism of acto-HMM NTP hydrolysis. Whereas all NTPs studied support force production and stiffness that vary by a factor 2 or less, the unloaded shortening velocity ( V u) of muscle fibers varies by almost 10-fold. 2-Deoxy ATP (dATP) was unique in that V u was 30% greater than with ATP. Parallel behavior was observed between V f and the steady-state maximum actin-activated HMM ATPase rate. Further comparisons suggest that the variation in force correlates with the rate and equilibrium constant for NTP cleavage; the variations in V u or V f are related to the rate of cross-bridge dissociation caused by NTP binding or to the rate(s) of product release.

Enzymes harboring unnatural amino acids: mechanistic and structural analysis of the enhanced catalytic activity of a glutathione transferase containing 5-fluorotryptophan.

The catalytic characteristics and structure of the M1-1 isoenzyme of rat glutathione (GSH) transferase in which all four tryptophan residues in each monomer are replaced with 5-fluorotryptophan are described. The fluorine-for-hydrogen substitution does not change the interaction of the enzyme with GSH even though two tryptophan residues (Trp7 and Trp45) are involved in direct hydrogen-bonding interactions with the substrate. The rate constants for association and dissociation of the peptide, measured by stopped-flow spectrometry, remain unchanged by the unnatural amino acid. The 5-FTrp-substituted enzyme exhibits a kcat of 73 s-1 as compared to 18 s-1 for the native enzyme toward 1-chloro-2,4-dinitrobenzene. That the increase in the turnover number is due to an enhanced rate of product release in the mutant is confirmed by the kinetics of the approach to equilibrium for binding of the product. The crystal structure of the 5-FTrp-containing enzyme was solved at a resolution of 2.0 A by difference Fourier techniques. The structure reveals local conformational changes in the structural elements that define the approach to the active site which are attributed to steric interactions of the fluorine atoms associated with 5-FTrp146 and 5-FTrp214 in domain II. These changes appear to result in the enhanced rate of product release. This structure represents the first of a protein substituted with 5-fluorotryptophan.

Kinetics of methylation and binding of DNA by the EcoRV adenine-N6 methyltransferase

The EcoRV DNA methyltransferase (M.EcoRV) specifically methylates the first adenine within its recognition sequence GATATC. Methylation rates of DNA by this enzyme are strongly influenced by the length of oligonucleotide substrates employed. If substrates > 20 bp compared to a 12mer substrate, the kcat/Kmincreases 100-fold, although the enzyme does not contact more than 12 base-pairs on the DNA. Single-turnover rates are higher than kcatvalues. M.EcoRV binding to DNA is fast but dissociation from the DNA is slow, demonstrating that the multiple-turnover rate is limited by the rate of product release. The kinetics of DNA binding by M.EcoRV are not in accordance with the thermodynamics binding constant, suggesting that the M.EcoRV-DNA complex is involved in a slow conformational change. The salt dependence of DNA binding is different for non-specific substrates (d ln(KAss)/d ln(cNaCl) = −2, indicative of electrostatic interactions) and specific substrates (d ln(KAss)/d ln(cNaCl) = +1, indicative of hydrophobic interactions). This result demonstrates that the M.EcoRV-DNA complex has a different conformation in both binding modes. M.EcoRV does not discriminate between hemimethylated and unmethylated substrates. Using the 20mer we have analyzed the temperature and pH dependence of the single-turnover rate constant of M.EcoRV-DNA methylation by M.EcoRV has an activation energy of 40 kJ/mol and its rate increases with increasing pH. The pH dependence reveals the presence of an ionizable residue with a pKaof 7.9, which must be unprotonated for catalysis. The rates of DNA methylation remain unchanged if an abasic site is introduced instead of the thymidine residue that is base-paired to the target adenine, demonstrating that flipping out the target adenine cannot contribute to the rate-limiting step of the enzymatic reaction.

Unisite ATP hydrolysis by soluble Rhodospirillum rubrum F 1–ATPase is accelerated by Ca 2+

At saturating concentrations of ATP, soluble F 1 from the Rhodospirillum rubrum (RF 1) exhibits a higher rate of hydrolysis with Ca 2+ than with Mg 2+. The mechanisms involved in the expression of a higher catalytic activity with Ca 2+ were explored by measuring the ATPase activity of RF 1 at substiochiometric concentrations of ATP (unisite conditions). At a ratio of 0.25 [γ- 32 P ]ATP per RF 1, the enzyme exhibited a 50 times higher hydrolytic rate with Ca 2+ than with Mg 2+. The rate of [γ- 32 P ]ATP binding to RF 1 was in the same range with the two divalent metal ions. Centrifugation–filtration of RF 1 exposed to substoichiometric [γ- 32 P ]ATP concentrations and Mg 2+ through Sephadex columns yielded an enzyme that contained [γ- 32 P ]ATP and [ 32 P ]phosphate in a stoichiometry that was close to one. In the presence of Ca 2+, the eluted enzyme did not contain [γ- 32 P ]ATP nor [ 32 P ]phosphate. This indicated that the rate of product release was faster with Ca 2+ than with Mg 2+. It was also observed that the ratio of multisite to unisite hydrolysis rates was of similar magnitude with both divalent cations. This suggests that they do not affect differently the cooperative mechanisms that may exist between catalytic sites. In consequence, the higher ATPase activity of RF 1 in presence of Ca 2+ strongly suggests that the retention time of products is decreased in the presence of this cation.

Nucleotide-depleted Beef Heart F1-ATPase Exhibits Strong Positive Catalytic Cooperativity

Catalytic cooperativity is a central feature of the binding change mechanism for F0F1-ATP synthases. However, in a recent publication (Reynafarje, B. D., and Pedersen, P. L. (1996) J. Biol. Chem. 271, 32546-32550), Reynafarje and Pedersen claim that cooperative effects are an artifact caused by endogenous nucleotides and that when such nucleotides are removed, the multiple catalytic sites on MF1 behave independently during ATP hydrolysis. In contrast to this conclusion, we show here that when ATP is loaded at a single catalytic site on nucleotide-depleted MF1, the rate of product release is accelerated by up to 5 x 10(4)-fold by the binding of ATP at adjacent catalytic sites. Hence, nucleotide-depleted MF1 is not an exception but does in fact show strong cooperative interactions. In addition, evidence is presented supporting a random order for product release during ATP hydrolysis.

Genetic analysis of the Chlamydomonas reinhardtii I-CreI mobile intron homing system in Escherichia coli.

We have developed and used a genetic selection system in Escherichia coli to study functional requirements for homing site recognition and cleavage by a representative eukaryotic mobile intron endonuclease. The homing endonuclease, I-CreI, was originally isolated from the chloroplast of the unicellular green alga Chlamydomonas reinhardtii. I-CreI homing site mutants contained base pair substitutions or single base deletions that altered the rate of homing site cleavage and/or product release. I-CreI endonuclease mutants fell into six phenotypic classes that differed in in vivo activity, toxicity or genetic dominance. Inactivating mutations clustered in the N-terminal 60% of the I-CreI amino acid sequence, and two frameshift mutations were isolated that resulted in premature translation termination though retained partial activity. These mutations indicate that the N-terminal two-thirds of the I-CreI endonuclease is sufficient for homing site recognition and cleavage. Substitution mutations altered in four potential active site residues were examined: D20N, Q47H or R70A substitutions inactivated endonuclease activity, whereas S22A did not. The genetic approach we have taken complements phylogenetic and structural studies of mobile intron endonucleases and has provided new information on the mechanistic basis of I-CreI homing site recognition and cleavage.

Kinetic Mechanism of Monomeric Non-claret Disjunctional Protein (Ncd) ATPase

The non-claret disjunctional protein (Ncd) is a kinesin-related microtubule motor that moves toward the negative end of microtubules. The kinetic mechanism of the monomer motor domain, residues 335-700, satisfied a simple scheme for the binding of 2'-3'-O-(N-methylanthraniloyl) (MANT) ATP, the hydrolysis step, and the binding and release of MANT ADP, where T, D, and Pi refer to nucleotide triphosphate, nucleotide diphosphate, and inorganic phosphate, respectively, and MtN is the complex of an Ncd motor domain with a microtubule site. Rate constants k1 and k-4 are the rates of a first order step, an isomerization induced by nucleotide binding. The apparent second order rate constants for the binding steps are 1.5 x 10(6) M-1 s-1 for MANT ATP and 3.5 x 10(6) M-1 s-1 for MANT ADP (conditions, 50 mM NaCl, pH 6.9, 21 degrees C). The rate constant of the hydrolysis step (k2) was obtained from quench flow measurements of the phosphate burst phase corrected for the contribution of the rate of product release to the transient rate constant. The rate of phosphate dissociation was not measured; the value was assigned to account for a steady state rate of 3 s-1. The MtN complex is dissociated by ATP at a rate of 10 s-1 based on light scattering measurements. Dissociation constants of Ncd-nucleotide complexes from microtubules increased in the order adenosine 5'-O-(thiotriphosphate) (ATPgammaS) < ADP-AlF4 < ATP < ADP < ADP-vanadate. Comparison of the properties of Ncd with a monomeric kinesin K332 (Ma and Taylor (1997) J. Biol. Chem. 272, 717-723) showed a close similarity, except that the rate constants for the hydrolysis and ADP release steps and the steady state rate are approximately 15-20 times smaller for Ncd. There are two differences that may affect the reaction pathway. The rate of dissociation of MtN by ATP is comparable to the rate of the hydrolysis step, and N.T may dissociate in the cycle, whereas for kinesin, dissociation occurs after hydrolysis. The rate of dissociation of MtN by ADP is larger than the rate of ADP release from MtN.D, whereas for the microtubule-kinesin complex, the rate of dissociation by ADP is smaller than the rate of ADP release. The monomeric Mt.Ncd complex is not processive.

Oxidation Kinetics of Ethanol by Human Cytochrome P450 2E1: RATE-LIMITING PRODUCT RELEASE ACCOUNTS FOR EFFECTS OF ISOTOPIC HYDROGEN SUBSTITUTION AND CYTOCHROME b5 ON STEADY-STATE KINETICS

A number of cytochrome P450 (P450) 2E1 substrates are known to show kinetic deuterium isotope effects of approximately 5 on Km (DK = DKm/HKm), but not on kcat, in rat liver microsomes (e.g. N-nitrosodimethylamine, ethanol, and CH2Cl2). We observed DKm values of 3-5 for recombinant human P450 2E1-catalyzed ethanol oxidation. Replacing NADPH and O2 with the oxygen surrogate cumene hydroperoxide yielded similar results. Ferric P450 2E1 reduction was fast (k >1000 min-1) even in the absence of substrate. These results indicate that the basis for the increase in Km is in the latter portion of the catalytic cycle. The intrinsic isotope effect (Dk) for ethanol oxidation was determined (competitively) to be 3.8, indicating that C-H bond cleavage is isotopically sensitive. Pre-steady-state studies showed a burst of product formation (k = 410 min-1), with the burst amplitude corresponding to the P450 concentration. Deuteration of ethanol resulted in an isotope effect of 3.2 on the rate of the burst. We conclude that product release is rate-limiting in the oxidation of ethanol to acetaldehyde by P450 2E1. The steady-state kinetics can be described by a paradigm in which the kcat approximates the rate of product release, and Km is an expression in which the denominator is dominated by the rate of C-H bond breaking.

Effect of substrate concentration and nitrate inhibition on product release and heavy metal removal by aCitrobacter sp.

A Citrobacter sp. accumulates heavy metals as cell-bound metal phosphates, utilizing phosphate released by the enzymatic cleavage of a phosphomonoester substrate. The effect of increased substrate (glycerol 2-phosphate, G2P) concentration on phosphate release and heavy metal accumulation was evaluated using a stirred tank reactor (STR) and a plug flow reactor (PFR). A significant improvement in metal removal was achieved with increased substrate concentration using immobilized Citrobacter cells in the PFR, which was not observed using free cells in the STR. Nitrate is an inhibitor of the Citrobacter phosphatase. This inhibition was concentration dependent and reversible. The rate of product release was restored by increasing the concentration of substrate (G2P). The ratio of rates of phosphate release under two different conditions (different nitrate and G2P concentrations) can be described by a equation developed from Michaelis-Menten kinetics. The concentration of substrate required for restoration of maximum velocity, V(max), in a batch and continuous-flow system can be predicted by substitution and calculation; this was confirmed by an experiment in model systems using cell suspensions and polyacrylamide gel immobilized cells in a flow-though column. For use in industrial situations it may be uneconomical or infeasible to supply additional substrate. Bioreactor activity was also restored by increasing the flow residence time, in accordance with a Michaelis-Menten-based model to describe removal of lanthanum from nitrate-supplemented flow in a PFR.

Effect of substrate concentration and nitrate inhibition on product release and heavy metal removal by a Citrobacter sp.

A Citrobacter sp. accumulates heavy metals as cell-bound metal phosphates, utilizing phosphate released by the enzymatic cleavage of a phosphomonoester substrate. The effect of increased substrate (glycerol 2-phosphate, G2P) concentration on phosphate release and heavy metal accumulation was evaluated using a stirred tank reactor (STR) and a plug flow reactor (PFR). A significant improvement in metal removal was achieved with increased substrate concentration using immobilized Citrobacter cells in the PFR, which was not observed using free cells in the STR. Nitrate is an inhibitor of the Citrobacter phosphatase. This inhibition was concentration dependent and reversible. The rate of product release was restored by increasing the concentration of substrate (G2P). The ratio of rates of phosphate release under two different conditions (different nitrate and G2P concentrations) can be described by a equation developed from Michaelis–Menten kinetics. The concentration of substrate required for restoration of maximum velocity, Vmax, in a batch and continuous-flow system can be predicted by substitution and calculation; this was confirmed by an experiment in model systems using cell suspensions and polyacrylamide gel immobilized cells in a flow-though column. For use in industrial situations it may be uneconomical or infeasible to supply additional substrate. Bioreactor activity was also restored by increasing the flow residence time, in accordance with a Michaelis–Menten-based model to describe removal of lanthanum from nitrate-supplemented flow in a PFR. © 1997 John Wiley & Sons, Inc. Biotechnol Biotechnol Bioeng 55:821–830, 1997.