CRYSTALLINE TRYPSIN : V. KINETICS OF THE DIGESTION OF PROTEINS WITH CRUDE AND CRYSTALLINE TRYPSIN

The rate of digestion, as determined by the increase in non-protein nitrogen or formol titration, of casein, gelatin, and hemoglobin with crystalline trypsin preparations increases nearly in proportion to the concentration of protein, but with crude pancreatic extract the rate of digestion becomes independent of the protein concentration in concentrations of more than 2.5 per cent. With both enzymes the rate of digestion of mixtures of 5 per cent casein and gelatin is greater than would be expected from the point of view of a compound between enzyme and substrate. The rate of digestion of 5 per cent casein in the presence of 5 per cent gelatin is exactly the same as that of 5 per cent casein alone. This result is obtained with both enzymes. The digestion of casein with crude trypsin follows the course of a monomolecular reaction quite closely while with purified trypsin the velocity constant decreases as the reaction proceeds. In the case of hemoglobin the monomolecular velocity constant decreases with both purified and crude enzyme. When the reaction is followed by changes in the viscosity of the solution the abnormal effect of changing substrate concentration disappears and the reaction is in fair agreement with the monomolecular equation. The results as a whole indicate that the abnormalities of the reaction are due to the occurrence of several consecutive reactions rather than to the formation of a substrate enzyme compound.     

Enzymes and microemulsions. Activity and kinetic properties of liver alcohol dehydrogenase in ionic water-in-oil microemulsions

The activity and the kinetic properties of horse liver alcohol dehydrogenase have been studied in water-in-oil microemulsions containing sodium dodecyl sulfate (SDS) or hexadecyl trimethylammonium bromide (CTAB), 1-butanol or 1-pentanol or 1-hexanol or t-butanol, water and cyclohexane alone or with octane. In the anionic microemulsions (i.e. containing sodium dodecyl sulfate), the enzyme quickly lost its activity, but was efficiently protected by the coenzyme and some adenine nucleotides. In the cationic microemulsions (i.e. containing hexadecyl trimethylammonium bromide), the enzyme activity was more stable and with higher alcohols was stable for at least 20 min. The Michaelis constant of NAD+ calculated with respect to the water content was nearly constant and higher than in water. The maximum velocity in anionic microemulsions depends on the water content whereas in cationic microemulsions, the maximum velocity did not show a clear dependence on the water content and was close to the maximum velocity found in water. The pH dependence of Km and Vmax in these microemulsions was similar to that observed in water. The kinetic data for a hydrophobic substrate, cinnamyl alcohol, showed that this alcohol partitions between the pseudo-phases and thus the apparent Michaelis constant and the concentration at which substrate-excess inhibition appeared were increased. The catalytic properties of the enzyme in microemulsions were illustrated by the preparative reduction of cinnamaldehyde with cofactor recycling. The rate determination of NAD+ reduction and of 1-butanol/cinnamaldehyde redox reaction showed that at low water content (2.8%), the NAD+ reduction rate was close to zero whereas the redox reaction rate was about half of the rate at higher water content. Probably at low water content the coenzyme binding-dissociation rates are reduced much more than the binding-dissociation rates of the substrates and the rates of the ternary complex interconversion. The cationic microemulsions seemed to be very favorable medium for enzyme activity, the tetraalkyl ammonium surfactant causing less denaturation than the anionic detergent dodecyl sulfate.     

Spectral and kinetic studies on Pseudomonas L-phenylalanine oxidase (deaminating and decarboxylating)

Pseudomonas L-phenylalanine oxidase (deaminating and decarboxylating) contains two FAD molecules in one molecule of the enzyme (Koyama, H. (1983) J. Biochem. 93, 1313-1319). When the enzyme was mixed anaerobically with L-phenylalanine, beta-2-thienylalanine, L-tyrosine, or L-methionine, a spectral species (purple intermediate) with a broad absorption band around 540 nm was observed with each substrate, and decayed slowly. From the data on the overall reaction kinetics, the rate of the L-phenylalanine oxidase reaction was expressed as follows. e/v = e/Vm + A/[S] + B/[O2] where e represents the concentration of enzyme unit, v the rate of the overall reaction, Vm the maximum velocity, and A and B are constants. Furthermore, the reactions of the enzyme with beta-2-thienylalanine (mostly an oxygenase substrate) and L-methionine (an oxidase substrate) were analyzed by the "stopped flow" method. The following scheme for the mechanism of L-phenylalanine oxidase reaction with both substrates is proposed, based on the data obtained. (formula; see text) Where Eox represents the oxidized form of the enzyme unit, EoxS the enzyme unit (oxidized form)-substrate compound, X the purple intermediate with a characteristic broad absorption band around 540 nm, S the substrate and P the product.     

DOES THE KINETICS OF TRYPSIN DIGESTION DEPEND ON THE FORMATION OF A COMPOUND BETWEEN ENZYME AND SUBSTRATE

1. The velocity of hydrolysis of gelatin by trypsin increases more slowly than the gelatin concentration and finally becomes nearly independent of the gelatin concentration. The relative velocity of hydrolysis of any two substrate concentrations is independent of the quantity of enzyme used to make the comparison. 2. The rate of hydrolysis is independent of the viscosity of the solution. 3. The percentage retardation of the rate of hydrolysis by inhibiting substances, is independent of the substrate concentration. 4. There is experimental evidence that the enzyme and inhibiting substance are combined to form a widely dissociated compound. 5. If the substrate were also combined with the enzyme, an increase in the substrate concentration should affect the equilibrium between the enzyme and the inhibiting substance. This is not the case. 6. The rate of digestion of a mixture of casein and gelatin is equal to the sum of the rates of hydrolysis of the two substances alone, as it should be if the rate is proportional to the concentration of free enzyme. This contradicts the saturation hypothesis. 7. If the reaction is followed by determining directly the change in the substrate concentration, it is found that this change agrees with the law of mass action; i.e., the rate of digestion is proportional to the substrate concentration.     

Substrate-velocity relationships for the Trichoderma viride cellulase-catalyzed hydrolysis of cellulose

The influence of substrate and enzyme concentrations on the rate of saccharification of two defined insoluble cellulose substrates, Avicel (FMC Corp., Philadelphia, Pa.) and Solka-Floc (James River Co., Berlin, N.H.), by the cellulase enzyme system of Trichoderma viride was evaluated. In the assays, enzyme concentrations ranging from 0.004 to 0.016 IU/ml and substrate concentrations up to 10% (wt/vol) were used. Analysis by initial velocity methods found the maximum velocity of saccharification to be nearly equivalent for the two substrates and the Km for the two substrates to be of a similar magnitude, i.e., 0.20% (wt/vol) for Solka-Floc and 0.63% (wt/vol) for Avicel. Studies in which relatively high substrate concentrations (greater than 15 times the Km) were used demonstrated that the enzyme exhibited very different apparent substrate inhibition properties for the two substrates. The rate of saccharification of Avicel at relatively high substrate concentrations was up to 35% lower than the maximum rate which was observed at lower substrate concentrations. The Avicel concentration corresponding to the maximum rate of saccharification was dependent on the enzyme concentration. In contrast to the results with Avicel, the enzyme did not exhibit substrate inhibition with the Solka-Floc substrate. Potential differences in the degree of substrate inhibition with different substrates, as reported here, are particularly relevant to the experimental design of comparative studies.     

Substrate-velocity relationships for the Trichoderma viride cellulase-catalyzed hydrolysis of cellulose.

The influence of substrate and enzyme concentrations on the rate of saccharification of two defined insoluble cellulose substrates, Avicel (FMC Corp., Philadelphia, Pa.) and Solka-Floc (James River Co., Berlin, N.H.), by the cellulase enzyme system of Trichoderma viride was evaluated. In the assays, enzyme concentrations ranging from 0.004 to 0.016 IU/ml and substrate concentrations up to 10% (wt/vol) were used. Analysis by initial velocity methods found the maximum velocity of saccharification to be nearly equivalent for the two substrates and the Km for the two substrates to be of a similar magnitude, i.e., 0.20% (wt/vol) for Solka-Floc and 0.63% (wt/vol) for Avicel. Studies in which relatively high substrate concentrations (greater than 15 times the Km) were used demonstrated that the enzyme exhibited very different apparent substrate inhibition properties for the two substrates. The rate of saccharification of Avicel at relatively high substrate concentrations was up to 35% lower than the maximum rate which was observed at lower substrate concentrations. The Avicel concentration corresponding to the maximum rate of saccharification was dependent on the enzyme concentration. In contrast to the results with Avicel, the enzyme did not exhibit substrate inhibition with the Solka-Floc substrate. Potential differences in the degree of substrate inhibition with different substrates, as reported here, are particularly relevant to the experimental design of comparative studies.     

Chemical modification of adenylosuccinate synthetase from Escherichia coli by pyridoxal 5'-phosphate. Identification of an active site lysyl residue

Incubation of adenylosuccinate synthetase from Escherichia coli with low concentrations of pyridoxal 5'-phosphate (PLP) resulted in a rapid loss of activity (92%), concomitant with the formation of a Schiff base. The inactivation of the enzyme by PLP is apparently first order with respect to PLP. The pseudo-first order rate constant, Kapp, showed a hyperbolic dependence on the concentration of PLP, indicating that a kinetically significant PLP.enzyme intermediate is formed during the inactivation process. Stoichiometry and peptide isolation studies showed that 2 lysine residues were modified during reaction of the enzyme with PLP. The three substrates of adenylosuccinate synthetase (GTP, IMP, and aspartate) showed different effects in their ability to protect the enzyme against PLP inactivation. Complete protection of the enzyme against inactivation can be observed only in the presence of high concentrations of GTP. One lysine residue was protected under these conditions. In contrast to GTP, addition of the other two substrates either alone or together to reaction mixtures did not render protection. Peptide mapping by digesting the enzyme with trypsin revealed that the lysine shielded by GTP is Lys140. Replacing the Lys140 with Ile140 by site-directed mutagenesis resulted in total loss of the activity. These results suggest that Lys140 may play an important role in enzymatic activity.     

Use of Firefly Luciferase for ATP Measurement: Other Nucleotides Enhance Turnover

Firefly luciferase utilizes only ATP and a few closely related nucleotides as substrates for the formation of luciferyl adenylate which is an intermediate in the bioluminescent reaction sequence that oxidizes firefly luciferin. The enzyme shows two different time courses of light production depending on ATP concentration used: a flash with high concentrations of ATP (>8μM) or a fairly constant production of light with lower concentrations of ATP (< 1 μM). Many nucleotides, nucleotide-containing substances and other compounds, when added either prior to or 1 min after the addition of ATP, change the time course of light production. When added before ATP, these compounds yield a reaction mixture in which light production is fairly constant (at the level characteristic of the flash observed with that ATP concentration). When the compounds are added after ATP addition, light production is markedly stimulated and the higher rate of light production is maintained for several minutes. There is an increase in quanta of light produced per luciferase dimer from 1 to 5/min with the addition of any of several nucleotide analogues. These results are consistent with a stimulated release of the inhibitory product oxyluciferin, allowing turnover of the enzyme. This enzyme turnover permits more light output at high ATP concentrations, thus enhancing the sensitivity of enzyme determination.     

Kinetic studies of alkyl-dihydroxyacetone-phosphate (alkyl-glycerone-phosphate) synthase in peroxisomes of rat liver

The properties of peroxisomal enzyme alkylglycerone-phosphate synthase were studied in highly purified peroxisome fractions of rat liver. The requirements for optimal enzyme activity: pH and composition of the reaction mixture, incubation time, and enzyme concentration were investigated, and kinetic studies performed employing both different long-chain fatty alcohols and acyl dihydroxyacetone phosphates as substrates. Activities of the synthase considerably higher as reported before were found in the peroxisome preparation, with alkylglycerone (alkyldihydroxyacetone) phosphate as the sole product of the exchange reaction. The kinetic studies revealed divergent properties of peroxisomal synthase with respect to the substrates involved. Whereas the substrate concentration versus reaction velocity plot for the fatty alcohols reflects Michaelis-Menten kinetic behavior, it displays a maximum followed by inhibition with regard to the acylglycerone phosphate. The enzyme accepts different acylglycerone phosphates without much specificity but it is most active with 9-cis-octadecenol.     

Synergistic substrates determination with biosensors

High sensitive biosensors for heterocyclic compounds determination were built using oxidases-catalyzed hexacyanoferrate(III) reduction in the presence of these compounds. As oxidases Aspergillus niger glucose oxidase and recombinant Microdochium nivale carbohydrate oxidase were used. The biosensors were build using graphite electrodes and entrapped solution of the oxidases. The sensitivity of the biosensors achieves 5.2-14.5 microA microM-1 cm-2. The detection limit of some heterocyclic compounds was 0.2 microM. The sensitivity of biosensors was 300-10,000 times larger in comparison to hexacyanoferrate(III). To background the scheme of biosensors action kinetics of synergistic substrates oxidation was investigated in homogenous solution. The measurements showed that the rate of the reduction of low reactive substrate (hexacyanoferrate(III)) increased due to synergistic action of high reactive substrates (oxidized heterocyclic compounds). The modeling revealed the limiting step of the process. The increase of hexacyanoferrate(III) reduction rate is determined by the rate of reduced enzymes interaction with oxidized heterocyclic compound. The oxidation of heterocyclic compounds (mediators) with hexacyanoferrate(III) does not limit the process. The analysis of macrokinetics of biosensors action showed that synergistic effect may be realized and high biosensors sensitivity may be achieved if diffusion module of the enzyme reaction with the oxidized mediator and of a cross reaction is larger than 0.5. The calculated relative sensitivity is about three times higher in comparison to experimentally determined that may be caused by the limited stability of oxidized heterocyclic compounds and/or some external diffusion limitation of substrates.     

Validity of quasi-steady-state and transfer-function representations for input-output relation in a Michaelis-Menten reaction

In relation to the input-output characteristics of enzymatic reactions in the cellular metabolism and biochemical reactors, the validity of the quasi-steady-state and transfer-function representations of reaction velocity has been examined for a basic Michaelis-Menten reaction employing computer simulation, that is, numerical integration of the rate equation. The well-known S-v relationship (relationship between substrate concentration and reaction velocity)derived on the quasi-steady-state assumption is found to be in general a good approximation to the actual velocity throughout the temporal progress of the reaction. The validity of the approximation depends on a ratio of the Michaelis constant to the total enzyme concentration in the reaction system rather than on the individual rate constants. A transfer-function representation is derived on assuming an exponential change in the reaction velocity for the indicial response to the substrate influx rate. The representation has a wider valid region with a decrease in influx rate than with an increase in the influx rate. The validity is most dependent on a ratio of total enzyme concentration to the steady-state concentration of the substrate. The analysis of the linear sensitivity of the reaction velocity to rate constants reveals that the characteristics of these valid representations in systems analysis change according to the phase of the reaction.     

Affinity labeling of the inhibitory DPNH site of bovine liver glutamate dehydrogenase by 5'-fluorosulfonylbenzoyl adenosine.

A new adenosine analogue has been synthesized, 5'-fluorosulfonylbenzoyl adenosine, which reacts covalently with bovine liver glutamate dehydrogenase with the incorporation of approximately 1 mol of 5'-sulfonylbenzoyl adenosine per peptide chain. Native glutamate dehydrogenase is known to be inhibited by relatively high concentrations of DPNH by binding to a second noncatalytic site; the major change in the kinetic characteristics of the modified enzyme is a total loss of this inhibition by DPNH. The modified enzyme retains full catalytic activity as measured in the absence of allosteric ligands, is still inhibited more than 90% by GTP, and is activated normally by ADP. These results demonstrate that the catalytic as well as the GTP and ADP regulatory sites are distinct from the inhibitory DPNH site. The rate constant for reaction of 5'-fluorosulfonylbenzoyl adenosine is decreased by high concentrations of DPNH alone or by DPNH plus GTP, but not by the substrate alpha-ketoglutarate, the coenzymes DPN or TPNH, or the regulators ADP or GTP alone. These observations are consistent with the postulate that the 5'-fluorosulfonylbenzoyl adenosine attacks exclusively the second inhibitory DPNH site. The DPNH inhibition is abolished when an average of only 0.5 mol of 5'-sulfonylbenzoyl adenosine per peptide chain has been incorporated. The structure of 5'-fluorosulfonylbenzoyl adenosine is critical in determining the course of the modification reaction. The smaller compound p-fluorosulfonylbenzoic acid does not affect the kinetic characteristics of the enzyme, and the isomeric compound 3'-fluorosulfonylbenzoyl adenosine produces a different pattern of changes in the regulatory properties (Pal. P. K., Wechter, W. J., and Colman, R. F. (1975) Biochemistry 14, 707-715). Indeed, enzyme which has combined stoichiometrically with 5'-fluorosulfonylbenzoyl adenosine is still able to react with 3'-fluorosulfonylbenzoyl adenosine; thus, the two adenosine analogues appear to react at distinct sites on glutamate dehydrogenase. It is proposed that 5'-fluorosulfonylbenzoyl adenosine will be complementary to 3'-fluorosulfonylbenzoyl adenosine as a general affinity label for dehydrogenases as well as other classes of enzymes which use adenine nucleotides as substrates or regulators.     

Diffusional effects on the heterogeneous kinetics of two-substrate enzymic reactions.

When a two-substrate reaction is catalyzed by a surface bound enzyme, the diffusion of both substrates can considerably modify the kinetic properties of the reaction. According to this theoretical analysis, limitations in substrate diffusion yield very different effects depending whether the two substrates have similar or different affinities for the enzyme. With two substrates of comparable affinities the diffusion of the two substrates can be limiting, and similar activity dependences on the two substrate concentrations are obtained. Under these conditions, diffusional limitations may only slightly influence half-maximal-activity substrate concentrations. With two substrates of widely different affinities, on the contrary, the rate of the enzymic reaction can only be limited by the diffusion of the high-affinity substrate, used at the lower concentration. Under these conditions, in the presence of diffusional limitations the activity dependence on the two substrate concentrations are highly different, and the half-maximal-activity concentration is increased and decreased for the high- and low-affinity substrates, respectively. The theoretical results are verified by experimental data previously obtained with collagen-bound aspartate aminotransferase and sorbitol dehydrogenase.     

Some examples of the use of computer-produced contour plots in the fitting of enzyme rate equations to reaction-velocity measurements

The use of computer-based isotach plots, relating reaction velocity to simultaneous variation of two substrates or effectors of an enzyme, in producing estimates of the parameters of enzyme rate equations was investigated. The computer program (;SYMAP') incorporates an interpolation algorithm, and the superiority of this over visual estimation in producing interpolated velocity values for the estimation of parameter values by conventional double-reciprocal plots is described. The usefulness of the SYMAP program in monitoring the process of fitting data obtained by simultaneous changes in two experimental variables is also described. It is shown that if the residual errors are weighted by a procedure described elsewhere (Ottaway, 1971b, 1973), the percentage error of the computed velocity is distributed evenly over a plot which contains a 100-fold variation in the concentration of one substrate and a 500-fold variation in the concentration of Mg(2+), and in which the velocity of the reaction (that catalysed by NAD kinase) varies over a 60-fold range. The two-dimensional percentage error plot was used to assess the limits within which an incomplete inhibition equation is valid, and to detect a discrepancy in an expected good fit, caused by an impurity in one of the substrates.     

Glucoheptonic acid derivative as a new transgalactosylation product

Transgalactosylation is increasingly used for modification of selected compounds because it may introduce new bioactive properties or improve existing ones. This paper presents the application of transgalactosylation activity of Kluyveromyces lactis β-galactosidase (EC 3.2.1.23) for a new derivative of glucoheptonic acid synthesis. The study concerned the impact of following factors on the course of reaction: content and ratio of substrates, enzyme dose, pH of the solution and the presence of salts. In the most favourable conditions (without salt), the final product concentration reached 54.5?g/L, which corresponded to 10.9% of dry matter. Research has shown that higher initial dry matter content results in higher product content (as % dm). The addition of 0.5–0.75?M MgCl₂ or 1?M NaCl led to significantly increased yield. In contrast, the presence of MnCl₂ or the lowest enzyme dose seemed to slow down the synthesis process. Increasing the pH over the optimal value for hydrolytic activity of β-galactosidase caused inhibition of transgalactosylation reaction. The molar ratio of 1.9:1 (sodium glucoheptonate:lactose) was the best among tested options. The described method allowed to successfully obtain a new compound with satisfactory yield in comparison to other transgalactosylation products.     

Steady-state enzyme kinetics in the Escherichia coli periplasm: a model of a whole cell biocatalyst.

This study provided analysis of in vivo enzyme kinetics in a model system which consisted of alkaline phosphatase in the periplasm of Escherichia coli. Modeling of complete substrate titration curves was achieved for a wide range of intraperiplasmic enzyme levels and outer membrane permeabilities. The results helped to identify the features most important to optimize in vivo reaction velocity. For many situations, a surprising finding was that maximum enzyme expression was not a major concern. For example, for moderate enzyme expression levels and moderate substrate levels (ca 0-5 mM), the limiting step for the enzyme in the periplasm was substrate (para-nitrophenylphosphate) diffusion through the outer membrane. In vivo reaction velocity was directly proportional to substrate concentration, outer membrane permeability, and the cell concentration. Velocity was also quite insensitive to a potent inhibitor of the enzyme. Even though diffusion-limited, periplasmic reaction velocity was quite sensitive to temperature, suggesting that the conformation of porin proteins in the E. coli outer membrane governed the average size of the pore. This model system therefore defined important features of bacterial whole cell biocatalyst design, which may also apply to other reactors using intact cells as catalysts.     

Utilization of the inactivation rate of coenzyme A transferase by thiol reagents to determine properties of the enzyme-CoA intermediate.

The rate of inactivation of succinyl-CoA:3-ketoacid coenzyme A transferase by thiol reagents is increased 3 to 100 times by very low concentrations of acyl-CoA substrates. The same maximum inactivation rate is found with acetoacetyl-CoA and succinyl-CoA. The enhanced rate of inactivation is caused by the stoichiometric formation of the enzyme-CoA intermediate and an accompanying conformation change of the enzyme. The inactivation rate provides a simple assay for the amount of enzyme present as the enzyme-CoA intermediate, using only catalytic concentrations of enzyme. This technique has been utilized to measure (a) a rate constant for hydrolysis of the enzyme-CoA intermediate of 0.10 min-1 at pH 8.1; (b) a stoichiometry of two active sites per enzyme molecule; and (c) the equilibrium constants for formation of the enzyme-CoA intermediate from dilute solutions of substrates (and hence for the overall reaction) by determining the ratio of [enzyme-CoA]/[enzyme] in the presence of a series of substrate "buffers" at different ratios of [RCOO-]/[RCOSCoA]. As the total concentration of acyl-CoA and carbosylate substrates is increased, the inactivation rate is decreased. This indicates that the Michaelis complexes are protected against inactivation.     

Sugar derivatives as new 6-phosphogluconate dehydrogenase inhibitors selective for the parasite Trypanosoma brucei

Sugar derivatives mimicking compounds which take part in the catalysed reaction have been assayed as alternative substrates and/or competitive inhibitors of 6-phosphogluconate dehydrogenase from Trypanosoma brucei and sheep liver. Phosphonate analogues have been synthesised and the new compound 5-deoxy-5-phosphono-D-arabinonate shows good selectivity towards the parasite enzyme. A number of 4-carbon and 5-carbon aldonates are strong inhibitors of the parasite enzyme with K(i) values below the substrate K(m) and some acyl derivatives are also potent inhibitors. At least five of the compounds showing a significant selectivity for the parasite enzyme represent leads for trypanocidal drugs against this recently validated target.     

Purification and properties of mitochondrial phosphoenolpyruvate carboxykinase from liver of Squalus acanthias

Liver from Squalus acanthias (spiny dogfish), a representative elasmobranch, contains approximately 1.4 units (mumol/min) of phosphoenolpyruvate carboxykinase activity per gram and approximately 90% of the total units of activity are localized in the mitochondria. The mitochondrial phosphoenolpyruvate carboxykinase was isolated and characterized. The purified enzyme has properties generally similar to those found in mammalian and avian species. The enzyme has a molecular weight of approximately 70,000 and exists in a functional state as a monomer. The isolated enzyme displays a dual cation requirement (e.g., 6 mM Mg2+ and 10 microM Mn2+) for maximal activity; very little activity is observed when Mg2+ is present alone, and the maximal activity attained with Mn2+ alone (millimolar concentrations required) is significantly less than that observed under optimal conditions with both cations present. When assayed in the direction of oxalacetate formation there is a lag in product formation with time; the lag can be eliminated by the presence of 50 microM GTP (product). The Km for substrates is not affected by Mn2+ concentration, suggesting that the role of Mn2+ may not be related to substrate binding. The apparent Km for phosphoenolpyruvate (approximately 1 mM) is substantially higher than that reported for phosphoenolpyruvate carboxykinase from other species. The activity of phosphoenolpyruvate carboxykinase is increased 70% by physiological concentrations of urea. Maximal velocity of the reaction in the direction of oxalacetate formation is approximately half that of the reverse reaction.     

Interrogating the mechanism of a tight binding inhibitor of AIR carboxylase

The enzyme aminoimidazole ribonucleotide (AIR) carboxylase catalyzes the synthesis of the purine intermediate, 4-carboxy-5-aminoimidazole ribonucleotide (CAIR). Previously, we have shown that the compound 4-nitro-5-aminoimidazole ribonucleotide (NAIR) is a slow, tight binding inhibitor of the enzyme with a Ki of 0.34 nM. The structural attributes and the slow, tight binding characteristics of NAIR implicated this compound as a transition state or reactive intermediate analog. However, it is unclear what molecular features of NAIR contribute to the mimetic properties for either of the two proposed mechanisms of AIR carboxylase. In order to gain additional information regarding the mechanism for the potent inhibition of AIR carboxylase by NAIR, a series of heterocyclic analogs were prepared and evaluated. We find that all compounds are weaker inhibitors than NAIR and that CAIR analogs are not alternative substrates for the enzyme. Surprisingly, rather subtle changes in the structure of NAIR can lead to profound changes in binding affinity. Computational investigations of enzyme intermediates and these inhibitors reveal that NAIR displays an electrostatic potential surface similar to a proposed reaction intermediate. The result indicates that AIR carboxylase is likely sensitive to the electrostatic surface of reaction intermediates and thus compounds which mimic these surfaces should possess tight binding characteristics. Given the evolutionary relationship between AIR carboxylase and N⁵-CAIR mutase, we believe that this concept extends to the mutase enzyme as well. The implications of this hypothesis for the design of selective inhibitors of the N⁵-CAIR mutase are discussed.