Regulation of a metabolic system in vitro: synthesis of threonine from aspartic acid.

Six enzymes involved in the conversion of aspartate to threonine have been extracted from Escherichia coli and separated from each other. Two of these enzymes, aspartokinase and homoserine dehydrogenase, have also been partially purified from Rhodopseudomonas spheroides. In an attempt to determine whether small changes in the kinetic properties of individual enzymes are important to the regulation of metabolic flux through a coupled reaction system, the partially purified enzymes were recombined in a variety of ways under reaction conditions designed to resemble the in vivo situation. These conditions include: use of an entire metabolic system rather than a single reaction; high enzyme concentrations at the same relative concentrations as found in the cell; and low, steady-state concentrations of substrates and products. Metabolic flux was followed spectrophotometrically and the concentrations of aspartic semialdehyde, hemoserine, O-phosphohomoserine, and threonine were measured. The results indicate that the threonine concentration is of major importance in regulating metabolic flux by inhibiting aspartokinase, the first reaction in threonine in the pathway. When threonine-insensitive aspartokinases were used, concentrations reached higher levels and the rate of NADPH oxidation remained higher. The fact that neither aspartic semialdehyde nor homoserine accumulated as the threonine concentration increased and the lack of correlation between changes in metabolic flux and ADP/ATP or NADPH/NADP ratios indicate that more subtle forms of metabolic regulation, such as "reverse cascade", secondary feedback sites, or "energy charge", are of little regulatory importance in this isolated, metabolic system. The results also emphasize the need for caution in projecting in vivo control mechanisms from in vitro experiments.     

Kinetic, dynamic, and pathway studies of glycerol metabolism by Klebsiella pneumoniae in anaerobic continuous culture: III. Enzymes and fluxes of glycerol dissimilation and 1,3-propanediol formation

The initial steps of glycerol dissimilation and 1,3-propanediol (1, 3-PD) formation by Klebsiella pneumoniae anaerobically grown on glycerol were studied by quantifying the in vitro and in vivo activities of enzymes in continuous culture under conditions of steady state and oscillation and during transient phases. The enzymes studied included glycerol dehydrogenase (GDH), glycerol dehydratase (GDHt), and 1,3-propanediol oxidoreductase (PDOR). Three conclusions can be drawn from the steady-state results. First, glycerol concentration in the culture is a key parameter that inversely affects the in vitro activities (concentrations) of all three enzymes, but has a positive effect on their in vivo activities. Growth rate significantly affects the ratio of in vitro and in vivo enzyme activities under low glycerol concentrations, but not under glycerol excess. Second, whereas the flux through the oxidative pathway of glycerol dissimilation is governed mainly by the regulation of in vivo enzyme activity on a metabolic level, the flux through the reductive pathway is largely controlled by the synthesis of enzymes. Third, GDHt is a major rate-liming enzyme for the consumption of glycerol and the formation of 1,3-PD in K. pneumoniae at high glycerol concentrations. Results from oscillating cultures revealed that both in vitro and in vivo activities of the enzymes oscillated. The average values of the in vitro activities during an oscillation cycle agreed well with their corresponding values for nonoscillating cultures under similar environmental conditions. Experiments with step changes in the feed concentration of glycerol demonstrated that growth and product formation are very sensitive to changes of substrate concentration in the culture. This sensitivity is due to the dynamic responses of the genetic and metabolic networks. They should be considered when modeling the dynamics of the culture and attempting to improve the formation of 1,3-PD.     

Presence and comparison of angiotensin converting enzyme in commercial cell culture sera

This study was conducted to determine the presence of the angiotensin converting enzyme in commercial sera used in cell culture medium. The aim of the research was to bring the presence of proteinases (angiotensin converting enzyme) to cell culture users' knowledge and to give some data for solving problems about the development of peptides as useful drugs. The enzymes, purified from foetal bovine, adult bovine, foetal equine, adult equine, and human sera, showed molecular weights of about 170 kDa. Captopril and lisinopril inhibited enzyme activities at nanomolar concentrations. The enzymes were able to hydrolyze, with different efficiency, angiotensin I, bradykinin and epidermal mitosis inhibiting pentapeptide. The heat inactivation of commercial sera at 56 degrees C for 30 min showed a reduction of ACE activity of about 35-80%. Therefore, the presence of ACE activity in commercial sera can influence the activity of biological peptides tested on cell lines cultured "in vitro."     

Dissecting enzyme regulation by multiple allosteric effectors: nucleotide regulation of aspartate transcarbamoylase

The enzyme aspartate transcarbamoylase (ATCase, EC 2.1.3.2 of Escherichia coli), which catalyzes the committed step of pyrimidine biosynthesis, is allosterically regulated by all four ribonucleoside triphosphates (NTPs) in a nonlinear manner. Here, we dissect this regulation using the recently developed approach of random sampling-high-dimensional model representation (RS-HDMR). ATCase activity was measured in vitro at 300 random NTP concentration combinations, each involving (consistent with in vivo conditions) all four NTPs being present. These data were then used to derive a RS-HDMR model of ATCase activity over the full four-dimensional NTP space. The model accounted for 90% of the variance in the experimental data. Its main elements were positive ATCase regulation by ATP and negative by CTP, with the negative effects of CTP dominating the positive ones of ATP when both regulators were abundant (i.e., a negative cooperative effect of ATP x CTP). Strong sensitivity to both ATP and CTP concentrations occurred in their physiological concentration ranges. UTP had only a slight effect, and GTP had almost none. These findings support a predominant role of CTP and ATP in ATCase regulation. The general approach provides a new paradigm for dissecting multifactorial regulation of biological molecules and processes.     

Properties of a lipase produced by Beauveria bassiana: purification and biochemical studies

Lipases are some of the crucial enzymes during the fungal penetrating process of the insect integument. Due to the importance and lack of information on the microbial lipases of Beauveria bassiana, investigations were carried out to purify and biochemically characterize these enzymes. The results obtained on growth medium demonstrated the highest activity of lipase 6 days after inoculation while the pH of the medium was 7.1. After three purification steps, the purified enzyme was 9.91-fold with specific activity of 20816 U/mg protein, recovery of 25% and molecular weight of 25 kDa. The purified lipase had the optimal pH and temperature at 7 and 35°C and was stable for 36–72 h under those conditions. Ca^{2 +} significantly increased the enzyme activity and NaCl decreased it at all the tested concentrations. In addition, Mn^{2 +} had no effect on enzyme activity but Mg^{2 +} and Zn^{2 +} increased it only at the highest concentration used. Three out of the four inhibitors used, significantly decreased the purified lipase activity so that most inhibition and changes in the enzyme kinetic parameters were obtained by using different concentrations of EDTA. Knowledge of enzymology provides important information for the development of fungi as microbial pest control agents opening new avenues for study of the role of enzymes in virulence.     

Evolution of an antibiotic resistance enzyme constrained by stability and activity trade-offs

Pressured by antibiotic use, resistance enzymes have been evolving new activities. Does such evolution have a cost? To investigate this question at the molecular level, clinically isolated mutants of the beta-lactamase TEM-1 were studied. When purified, mutant enzymes had increased activity against cephalosporin antibiotics but lost both thermodynamic stability and kinetic activity against their ancestral targets, penicillins. The X-ray crystallographic structures of three mutant enzymes were determined. These structures suggest that activity gain and stability loss is related to an enlarged active site cavity in the mutant enzymes. In several clinically isolated mutant enzymes, a secondary substitution is observed far from the active site (Met182-->Thr). This substitution had little effect on enzyme activity but restored stability lost by substitutions near the active site. This regained stability conferred an advantage in vivo. This pattern of stability loss and restoration may be common in the evolution of new enzyme activity.     

Glucose-fructose oxidoreductase, a new enzyme isolated from Zymomonas mobilis that is responsible for sorbitol production.

The enzymes responsible for sorbitol formation in Zymomonas mobilis were investigated. A previously undescribed enzyme catalyzes the intermolecular oxidation-reduction of glucose and fructose to form gluconolactone and sorbitol. This enzyme has been purified; it had a subunit size of 40,000 daltons and is probably tetrameric at low pH. It contained tightly bound NADP as the hydrogen carrier and did not require any added cofactor for activity. In addition, a gluconolactonase has been isolated, although not completely purified. Together these two enzymes were capable of completely converting a 54% (wt/vol) equimolar mixture of glucose and fructose to sorbitol and sodium gluconate at the optimum pH of close to 6.2. The oxidoreductase had low affinities for its substrates, but natural environmental conditions would expose it to high concentrations of sugars. The amount of the enzyme in Z. mobilis cells was sufficient to account for the rate of sorbitol formation in vivo. However, the enzyme was present in the highest amounts when the cells were grown on glucose alone, and it was repressed by the presence of fructose; this was not the case with the gluconolactonase.     

Testing biochemistry revisited: how in vivo metabolism can be understood from in vitro enzyme kinetics

A decade ago, a team of biochemists including two of us, modeled yeast glycolysis and showed that one of the most studied biochemical pathways could not be quite understood in terms of the kinetic properties of the constituent enzymes as measured in cell extract. Moreover, when the same model was later applied to different experimental steady-state conditions, it often exhibited unrestrained metabolite accumulation.Here we resolve this issue by showing that the results of such ab initio modeling are improved substantially by (i) including appropriate allosteric regulation and (ii) measuring the enzyme kinetic parameters under conditions that resemble the intracellular environment. The following modifications proved crucial: (i) implementation of allosteric regulation of hexokinase and pyruvate kinase, (ii) implementation of V(max) values measured under conditions that resembled the yeast cytosol, and (iii) redetermination of the kinetic parameters of glyceraldehyde-3-phosphate dehydrogenase under physiological conditions.Model predictions and experiments were compared under five different conditions of yeast growth and starvation. When either the original model was used (which lacked important allosteric regulation), or the enzyme parameters were measured under conditions that were, as usual, optimal for high enzyme activity, fructose 1,6-bisphosphate and some other glycolytic intermediates tended to accumulate to unrealistically high concentrations. Combining all adjustments yielded an accurate correspondence between model and experiments for all five steady-state and dynamic conditions. This enhances our understanding of in vivo metabolism in terms of in vitro biochemistry.     

Stability of glucose-6-phosphate dehydrogenase in human cells cultivated in vitro]

It was shown that the thermal stability of glucose-6-phosphate dehydrogenase in human diploid cells is much higher than in human heteroploid cell lines HeLa and T-9. The purified enzymes from human diploid cells and from HeLa and T-9 cells possess similar thermal stabilities. Mixing of T-9 extracts with the purified enzyme preparations revealed that the non-stability factors of the dehydrogenase are present in the T-9 extracts. An addition of NADP- and NADPH-containing buffers and crystalline NADP to the heteroploid cell extracts stabilizes the enzyme. The thermal stability of the enzyme from "in vitro" cultivated human cells depends on the concentration of the coenzyme. It was also demonstrated that glucose-6-phosphate dehydrogenase stability in HeLa and T-9 extracts is the same at low concentrations of the coenzyme and after addition of crystalline NADP. However, at NADP concentration of 10(-3) M the enzyme stability in HeLa and T-9 extracts is different. It is assumed that the destabilizing factors are the enzymes possessing the nucleotidases activity, which is different in various cell lines.     

Yeast (Saccharomyces cerevisiae) fructose-1,6-bisphosphatase. Properties of phospho and dephospho forms and of two mutants in which serine 11 has been changed by site-directed mutagenesis

The properties of dephospho- and phosphofructose-1,6-bisphosphatase from the yeast Saccharomyces cerevisiae and of two mutant enzymes in which the phosphorylatable Ser11 had been changed by site-directed mutagenesis (Ser----Ala and Ser----Asp) were studied to clarify the role of cyclic AMP-dependent phosphorylation of yeast fructose-1,6-bisphosphatase. The mutant enzymes and wild type Ser11 fructose-1,6-bisphosphatase were overexpressed and purified to homogeneity. Phosphofructose-1,6-bisphosphatase was prepared by in vitro phosphorylation. The comparison of the properties of the above enzymes demonstrated that all four had similar maximum activity. However, the phosphoenzyme was about 3-fold more sensitive to AMP and fructose 2,6-bisphosphate inhibition than the dephosphoenzyme, suggesting that regulation operates in vivo by this mechanism, leading to decreased enzyme activity. The purified mutant enzymes Ala11 and Asp11 exhibited properties closely similar to those of dephospho- and phosphofructose-1,6-bisphosphatase, respectively. These results indicate that the functional group at residue 11 is an important factor in the regulation of fructose-1,6-bisphosphatase activity and that Ser(P) can be functionally substituted by Asp in this enzyme.     

Cadmium-dependent enzyme activity alteration is not imputable to lipid peroxidation

The effect of cadmium on the liver-specific activities of NADPH-cytochrome P450 reductase (CPR), malic dehydrogenase (MDH), glyceraldehyde-3-phosphate dehydrogenase (GADPH), and sorbitol dehydrogenase (SDH) was assessed 6, 24, and 48 h after administration of the metal to rats (2.5 mg/kg of body weight, as CdCl2, single ip injection). CPR specific activity increased after 6 h and afterward decreased significantly, while MDH specific activity increased up to 24 h and then remained unchanged. Both SDH and GADPH specific activities reduced after 6 h, the former only a little but the latter much more, and after 24 and 48 h were strongly inhibited. In vitro experiments, by incubating rat liver microsomes, mitochondria, or cytosol with CdCl2 in the pH range 6.0-8.0, excluded cadmium-induced lipid peroxidation as the cause of the reduction in enzyme activity. In addition, from these experiments, we obtained indications on the type of interactions between cadmium and the enzymes studied. In the case of CPR, the inhibitory effect is probably due to Cd2+ binding to the histidine residue of the apoenzyme, which, at physiological pH, acts as a nucleophilic group. In vitro, mitochondrial MDH was not significantly affected by cadmium at any pH, indicating that this enzyme is probably not involved in the decrease in mitochondrial respiration caused by this metal. As for GADPH specific activity, its inhibition at pH 7.4 and above is imputable to the binding of cadmium to the SH groups present in the enzyme active site, since in the presence of dithiothreitol this inhibition was removed. SDH was subjected to a dual effect when cytosol was exposed to cadmium. At pH 6.0 and 6.5, its activity was strongly stimulated up to 75 microM CdCl2 while at higher metal concentrations it was reduced. At pH 7.4 and 8.0, a stimulation up to 50 microM CdCl2 occurred but above this concentration, a reduction was found. These data seem to indicate that cadmium can bind to different enzyme sites. One, at low cadmium concentration, stimulates the SDH activity while the other, at higher metal concentrations, substitutes for zinc, thus causing inhibition. This last possibility seems to occur in vivo essentially at least 24 h after intoxication. The cadmium-induced alterations of the investigated enzymes are discussed in terms of the metabolic disorders produced which are responsible for several pathological conditions.     

Engineering productive enzyme confinement

Natural enzymes were selected to function inside the cell, not in the test tube; therefore, their performance is optimized for the crowded conditions encountered in vivo. Most man-made matrices for enzyme confinement lead to suboptimal catalytic activity. Ackerman and colleagues showed that an entrapping environment consisting of functionalized mesoporous silica actually enhances enzyme activity beyond the test-tube levels of free enzymes in solution. These findings provide an approach for dissecting the effect of various contributors to enzyme activity and thereby provide a means for fine-tuning the entrapping matrices to optimize enzyme performance in a rational way.     

Modulation of angiogenesis by tissue inhibitor of metalloproteinase-4

Despite the importance of MMP activity in the regulation of angiogenesis, relatively little is known about the role of TIMP-4, the most recently discovered endogenous MMP inhibitor, in modulating neovascularization. It has largely been assumed that all TIMPs are capable of inhibiting angiogenesis in vivo. However, it is now widely appreciated that TIMPs-1, -2, and -3 differ significantly in their ability to modulate angiogenic processes in vitro and angiogenesis in vivo. In order to study the effect of TIMP-4 in controlling angiogenesis, we have cloned and expressed TIMP-4 in a Pichia pastoris expression system, purified it to homogeneity, and tested its ability to regulate angiogenesis in vivo and in vitro. Our studies demonstrate that TIMP-4 is an inhibitor of capillary endothelial cell migration, but not of proliferation or of angiogenesis in vivo.     

Kinetic properties of enzyme populations in vivo: alkaline phosphatase of the Escherichia coli periplasm.

Studies were conducted to determine the role that diffusion may play in the in vivo kinetics of the Escherichia coli periplasmic enzyme, alkaline phosphatase (AP, encoded by the gene pho A). Passive diffusion of solutes, from solution into the periplasm, is thought to occur mainly through porins in the outer membrane. The outer membrane therefore serves as a diffusion barrier separating a population of periplasmic enzymes from bulk substrate. E. coli strains containing a plasmid with the pho A gene linked to the lac promoter were used in this study in order to vary the amount of enzyme per cell. Alkaline phosphatase assays were conducted with intact cells, and the substrate concentration at half-maximum velocity (normally the Km for the enzyme) was determined as a function of enzyme concentration per cell. The results showed that diffusion of substrate to the enzyme caused as much as a 1000-fold change in this parameter, compared to that of purified enzyme. This suggested that diffusion was the rate-limiting step of the enzymatic reaction in these cells. In agreement with this type of reaction, Eadie-Hofstee and Lineweaver-Burk plots were not linear. At their extremes, these plots represented two types of kinetics. At high substrate concentration, equilibrium of substrate between bulk solution and the periplasm was achieved, and the kinetic properties conformed to Michaelis-Menten. At low substrate concentrations, there were a large number of free (unbound) enzymes, and each substrate molecule that entered the periplasm, through the diffusion barrier, resulted in product formation.(ABSTRACT TRUNCATED AT 250 WORDS)     

High-throughput enzyme evolution in Saccharomyces cerevisiae using a synthetic RNA switch

Metabolic engineering can produce a wide range of bulk and fine chemicals using renewable resources. These approaches frequently require high levels of activity from multiple heterologous enzymes. Directed evolution techniques have been used to improve the activity of a wide range of enzymes but can be difficult to apply when the enzyme is used in whole cells. To address this limitation, we developed generalizable in vivo biosensors using engineered RNA switches to link metabolite concentrations and GFP expression levels in living cells. Using such a sensor, we quantitatively screened large enzyme libraries in high throughput based on fluorescence, either in clonal cultures or in single cells by fluorescence activated cell sorting (FACS). By iteratively screening libraries of a caffeine demethylase, we identified beneficial mutations that ultimately increased the enzyme activity in vivo by 33 fold and the product selectivity by 22 fold. As aptamer selection strategies allow RNA switches to be readily adapted to recognize new small molecules, these RNA-based screening techniques are applicable to a broad range of enzymes and metabolic pathways.     

Modulation of muscle phosphofructokinase at physiological concentration of enzyme

Two approaches have been used to study the allosteric modulation of phosphofructokinase at physiological concentration of enzyme; a "slow motion" approach based on the use of a very low Mg2+/ATP ratio to conveniently lower Vmax, and the addition of polyethylene glycol as a "crowding" agent to favor aggregation of diluted enzyme. At 0.6 mg/ml muscle phosphofructokinase exhibited a drastic decrease in the ATP inhibition and the concomitant increase in the apparent affinity for fructose-6-P, as compared to a 100-fold diluted enzyme. Similar results were obtained with diluted enzyme in the presence of 10% polyethylene glycol (Mr = 6000). Results with these two approaches in vitro were essentially similar to those previously observed in situ (Aragón, J. J., Felíu, F. E., Frenkel, R., and Sols, A. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 6324-6328), indicating that the enzyme is strongly dependent on homologous interactions at physiological concentrations. With polyethylene glycol it was observed that within the physiological range of concentration of substrates and the other positive effectors, fructose-2,6-P2 still activates the liver phosphofructokinase although it no longer significantly affects the muscle isozyme. In the presence of polyethylene glycol, muscle phosphofructokinase can approach its maximal rate even in the presence of physiologically high concentrations of ATP. Three minor activities of muscle phosphofructokinase have been studied at high enzyme concentration: the hydrolysis of MgATP (ATPase) and fructose-1,6-P2 (FBPase), produced in the absence of the other substrate, and the reverse reaction from MgADP and fructose-1,6-P2. The kinetic study of these activities has allowed a new insight into the mechanisms involved in the modulation of phosphofructokinase activity. The binding of (Mg)ATP at its regulatory site reduces the ability of the enzyme to cleave the bond of the terminal phosphate of MgATP at the substrate site. The positive effectors (Pi, cAMP, NH+4, fructose-1,6-P2, and fructose-2,6-P2) decrease the inhibitory effect of MgATP. Citrate and fructose-2,6-P2 both act as mechanistically "secondary" effectors in the sense that citrate does not inhibit and fructose-2,6-P2 does not activate the FBPase activity, requiring both the presence of ATP to affect the enzyme activity. In conclusion it appears that the regulatory behavior of mammalian phosphofructokinases is utterly dependent on the fact of their high concentrations in vivo.