The p-nitrophenyl phosphatase activity of muscle carbonic anhydrase

Carbonic anhydrase III from rabbit muscle, a newly discovered major isoenzyme of carbonic anhydrase, has been found to be also a p-nitrophenyl phosphatase, an activity which is not associated with carbonic anhydrases I and II. The p-nitrophenyl phosphatase activity has been shown to chromatograph with the CO₂ hydratase activity; both activities are associated with each of its sulfhydryl oxidation subforms; and both activities follow the same pattern of pH stability. This phosphomonoesterase activity of carbonic anhydrase III has an acidic pH optimum (<5.3); its true substrate appears to be the phosphomonoanion with a K_m of 2.8 mm. It is competitively inhibited by the typical acid phosphatase inhibitors phosphate (K_i = 1.22 × 10^?3M), arsenate (K_i = 1.17 × 10^?3M), and molybdate (K_i = 1.34 × 10^?7M), with these inhibitors having no effect on the CO₂ hydratase or the p-nitrophenyl acetate esterase activities of carbonic anhydrase III. The p-nitrophenyl acetate esterase activity of carbonic anhydrase III, on the other hand, has the sigmoidal pH profile with an inflection at neutral pH, typical of carbonic anhydrases for all of their substrates, and is inhibitable by acetazolamide (a highly specific carbonic anhydrase inhibitor) to the same degree as the CO₂ hydratase activity. The acid phosphatase-like activity of carbonic anhydrase III is slightly inhibited by acetazolamide at acidic pH, and inhibited to nearly the same degree at neutral pH. These data are taken to suggest that the phosphatase activity follows a mechanism different from that of the CO₂ hydratase and p-nitrophenyl acetate esterase activities and that there is some overlap of the binding sites.     

Inhibition by cupric ions of the hydration of CO2 catalyzed by carbonic anhydrase II

The inhibition by cupric ions of the hydration of CO₂ catalyzed by carbonic anhydrase II is interesting because of the results of Tuet al. obtained at chemical equilibrium, indicating that Cu²⁺ inhibits specifically a proton transfer in the catalytic pathway. We have measured this inhibition at steady state, using stopped-flow methods. The inhibition by Cu²⁺ of the hydration of CO₂ catalyzed by carbonic anhydrase II had aK _I near 1×10^?6 M atpH 7.0 and gave inhibition that is noncompetitive atpH 6.0 and mixed, but close to uncompetitive, atpH 6.8. ThepH dependence of this binding is consistent with a binding site for Cu²⁺ on the enzyme with apK _a near 7. The binding interaction between Cu²⁺ and the fluorescent inhibitor 5-dimethylaminonaphthalene-l-sulfonamide on carbonic anhydrase II was noncompetitive, indicating that the binding site for Cu²⁺ is distinct from the coordination sphere of zinc in which the actual interconversion of CO₂ and HCO ₃ ^? and the binding of sulfonamides takes place.     

Cloning,expression and some properties of α-carbonic anhydrase from Helicobacter pylori

The α-carbonic anhydrase gene from Helicobacter pylori strain 26695 has been cloned and sequenced. The full-length protein appears to be toxic to Escherichia coli, so we prepared a modified form of the gene lacking a part that presumably encodes a cleavable signal peptide. This truncated gene could be expressed in E. coli yielding an active enzyme comprising 229 amino acid residues. The amino acid sequence shows 36% identity with that of the enzyme from Neisseria gonorrhoeae and 28% with that of human carbonic anhydrase II. The H. pylori enzyme was purified by sulfonamide affinity chromatography and its circular dichroism spectrum and denaturation profile in guanidine hydrochloride have been measured. Kinetic parameters for CO₂ hydration catalyzed by the H. pylori enzyme at pH 8.9 and 25°C are k_cat=2.4×10⁵ s⁻¹, K_M=17 mM and k_cat/K_M=1.4×10⁷ M⁻¹ s⁻¹. The pH dependence of k_cat/K_M fits with a simple titration curve with pK_a=7.5. Thiocyanate yields an uncompetitive inhibition pattern at pH 9 indicating that the maximal rate of CO₂ hydration is limited by proton transfer between a zinc-bound water molecule and the reaction medium in analogy to other forms of the enzyme. The 4-nitrophenyl acetate hydrolase activity of the H. pylori enzyme is quite low with an apparent catalytic second-order rate constant, k_enz, of 24 M⁻¹ s⁻¹ at pH 8.8 and 25°C. However, with 2-nitrophenyl acetate as substrate a k_enz value of 665 M⁻¹ s⁻¹ was obtained under similar conditions.     

A modified pH drift assay for inorganic carbon accumulation and external carbonic anhydrase activity in microalgae

Threat of global warming due to carbon dioxide (CO₂) emissions has stimulated research into carbon sequestration and emissions reduction technologies. Alkaline scrubbing allows CO₂ to be captured as bicarbonate, which can be photochemically fixed by microalgae. The carbon concentrating mechanism (CCM), of which external carbonic anhydrase is a key component, allows organisms to utilise this bicarbonate. In order to select a suitable strain for this application, a screening tool is required. The current method for determining carbonic anhydrase activity, the Wilbur and Anderson assay, was found to be unsuitable as a screening tool as the associated error was unacceptably large and tests on whole cells were inconclusive. This paper presents the development of a new, whole cell assay to measure inorganic carbon uptake and external carbonic anhydrase activity, based on classical pH drift experiments. Spirulina platensis was successfully used to develop a correlation between the specific carbon uptake (C) and the specific pH change (dpH). The relationship is described by the following: C[mmol C (g dry algae)^?1?h^?1]?=?0.064?×?(dpH). Inhibitor and salt dissociation tests validated the activity and presence of external carbonic anhydrase and allowed correlation between the Wilbur and Anderson assay and the new whole cell assay. Screening tests were conducted on S. platensis, Scenedesmus sp., Chlorella vulgaris and Dunaliella salina that were found to have carbon uptake rates of 5.76, 5.86, 3.86 and 2.15 mmol C (g dry algae)^?1?h^?1, respectively. These results corresponded to the species' known bicarbonate utilisation abilities and validated the use of the assay as a screening tool.     

Oxygen-18 Exchange as a Measure of Accessibility of CO(2) and HCO(3) to Carbonic Anhydrase in Chlorella vulgaris (UTEX 263)

We have measured the exchange of ¹⁸O between CO₂ and H₂O in stirred suspensions of Chlorella vulgaris (UTEX 263) using a membrane inlet to a mass spectrometer. The depletion of ¹⁸O from CO₂ in the fluid outside the cells provides a method to study CO₂ and HCO₃⁻ kinetics in suspensions of algae that contain carbonic anhydrase since ¹⁸O loss to H₂O is catalyzed inside the cells but not in the external fluid. Low-CO₂ cells of Chlorella vulgaris (grown with air) were added to a solution containing ¹⁸O enriched CO₂ and HCO₃⁻ with 2 to 15 millimolar total inorganic carbon. The observed depletion of ¹⁸O from CO₂ was biphasic and the resulting ¹⁸C content of CO₂ was much less than the ¹⁸O content of HCO₃⁻ in the external solution. Analysis of the slopes showed that the Fick's law rate constant for entry of HCO₃⁻ into the cell was experimentally indistinguishable from zero (bicarbonate impermeable) with an upper limit of 3 × 10⁻⁴ s⁻¹ due to our experimental errors. The Fick's law rate constant for entry of CO₂ to the sites of intracellular carbonic anhydrase was large, 0.013 per second, but not as great as calculated for no membrane barrier to CO₂ flux (6 per second). The experimental value may be explained by a nonhomogeneous distribution of carbonic anhydrase in the cell (such as membrane-bound enzyme) or by a membrane barrier to CO₂ entry into the cell or both. The CO₂ hydration activity inside the cells was 160 times the uncatalyzed CO₂ hydration rate.     

Carbonic anhydrase catalyzed oxygen-18 exchange between bicarbonate and water

Oxygen-18 exchange techniques were applied to the dehydration of bicarbonate catalyzed by human carbonic anhydrase C. The rates of depletion of oxygen-18 from labeled bicarbonate were measured for both the catalyzed and uncatalyzed reactions at pH 9.4 and 25 °C. The equilibrium dissociation constant of the enzyme-substrate complex K is 0.321 ± 0.040 m and $\text{k}{}_{\mathrm{<mtext>enz</mtext>}}\text{=}\text{k}{}_2\text{K}{}_{\mathrm{m}}$ is (8.3 ± 1.9) × 10⁵m^?1 sec^?1 under these conditions. On the basis of these results it is demonstrated that the oxygen-18 exchange technique is capable of measuring K and k_enz for the carbonic anhydrase catalyzed dehydration of bicarbonate at a high pH range in which other kinetic techniques are not effective.It was also shown that the oxygen-18 exchange technique is an effective micromethod for the determination of carbonic anhydrase. Rates of isotopic depletion of labeled bicarbonate (in solutions of the enzyme) which fall outside the limits of error for the uncatalyzed rate of depletion demonstrate that this technique can detect concentrations of human carbonic anhydrase C as low as 5 × 10^?11m.     

Hydrolysis of p-nitrotrifluoroacetanilide catalyzed by water and imidazole

The hydrolyses of p-nitrotrifluoroacetanilide catalyzed by water and imidazole were examined at 70°C. The pH-rate constant profile of the hydrolysis in H₂O was examined in the pH range 0.0–11.4. The hydrolysis was independent of pH in the region from pH 1.0 to 4.5, presumably a water-catalyzed reaction. The rate constant and the D₂O solvent isotope effect for this reaction were 1.0 × 10^?4 sec^?1 and 3.7, respectively. Both natural imidazole and imidazolium cation catalyzed hydrolysis. The rate constant of the hydrolysis catalyzed by neutral imidazole was determined to be 5.4 × 10^?3M^?1 sec^?1 and the D₂O solvent isotope effect was 1.8.     

A study of the histidine residues of human carbonic anhydrase B using 270 MHz proton magnetic resonance

Nine resonances in the 270 MHz proton magnetic resonance spectrum of human carbonic anhydrase B have been identified with imidazole C₍₂₎ protons of histidine residues, six of which are observed to titrate with pK_a values in the range 4.7 to 7.4. The behaviour of the nine resonances has been studied in the presence of the inhibitors, iodide, cyanide, acetate, hexacyanochromate, and imidazole. Measurements have also been made of the enzyme in its apo, cobalt, and mono-alkylated forms. Used in conjunction with the crystal structure, these results have enabled the tentative assignment of all nine resonances to particular histidine residues in the amino-acid sequence. Three of the active-site histidines at positions 64, 67, and 200 have low pK_a values and cannot be directly linked to the activity of the enzyme. However, the resonances assigned to the three metal-liganding histidines do exhibit changes on anion binding and with pH, which parallel changes in the esterase activity. These results are consistent with the model of an ionizable water molecule bound to the zinc ion.Linewidth measurements of the resonances of the histidine residues on the enzyme surface are used to estimate pseudo-first-order rate constants of the order of 4 × 10³ s^?1 for D⁺ exchange between imidazole N and solvent in the absence of buffer. These rates are observed to increase in the presence of small amounts of the buffers Tris and imidazole.     

Carbonic anhydrase inhibitors: purification and inhibition studies of pigeon (Columba livia var. domestica) red blood cell carbonic anhydrase with sulfonamides

A carbonic anhydrase (CA, EC 4.2.1.1) from red blood cells of pigeons (Columba livia var. domestica), clCA, was purified to homogeneity. Its kinetic parameters for the CO₂ hydration reaction were measured. With a k_cat/K_m of 1.1?×?10⁸ M^?1 s^?1, and a k_cat of 1.3?×?10⁶ s^?1, clCA has a high activity, similar to that of the human isoform hCA II. A group of 25 aromatic/heterocyclic sulfonamides incorporating the sulfanilamide, homosulfanilamide, benzene-1,3-disulfonamide, and acetazolamide scaffolds showed variable inhibitory activity against the pigeon enzyme, with K_Is in the range of 1.9–3460?nM. Red blood cells of pigeons, like those of ostriches, contain thus just one CA isoform, unlike the blood of mammals, which normally contain two isoforms, one of low (CA I-like) and one of very high activity (CA II-like). However, from the sulfonamide inhibition viewpoint, the pigeon enzyme was more similar to hCA II than to the ostrich enzyme.     

Effect of pH on structure,function, and stability of mitochondrial carbonic anhydrase VA

Mitochondrial carbonic anhydrase VA (CAVA) catalyzes the hydration of carbon dioxide to produce proton and bicarbonate which is primarily expressed in the mitochondrial matrix of liver, and involved in numerous physiological processes including lipogenesis, insulin secretion from pancreatic cells, ureagenesis, gluconeogenesis, and neuronal transmission. To understand the effect of pH on the structure, function, and stability of CAVA, we employed spectroscopic techniques such as circular dichroism, fluorescence, and absorbance measurements in wide range of pH (from pH 2.0 to pH 11.5). CAVA showed an aggregation at acidic pH range from pH 2.0 to pH 5.0. However, it remains stable and maintains its secondary structure in the pH range, pH 7.0–pH 11.5. Furthermore, this enzyme has an appreciable activity at more than pH 7.0 (7.0 < pH ≤ 11.5) with maximum activity at pH 9.0. The maximal values of k_cat and k_cat/K_m at pH 9.0 are 3.7?×?10⁶ s^?1 and 5.5?×?10⁷ M^?1 s^?1, respectively. However, this enzyme loses its activity in the acidic pH range. We further performed 20-ns molecular dynamics simulation of CAVA to see the dynamics at different pH values. An excellent agreement was observed between in silico and in vitro studies. This study provides an insight into the activity of CAVA in the pH range of subcellular environment.     

On the Reaction of Superoxide With DMPO/*OOH

A kinetic model has been used to estimate the rate constant for the reaction of superoxide (O₂/OOH) with the superoxide spin adduct of 5.5-dimethylpyrroline-N-oxide. DMPO/OOH. This rate constant is estimated to be 4.9 (± 2.2) × 10⁶ M^?1 s^?1, pH 7.4 and 25°C.     

Cloning,expression and biochemical characterization of a β-carbonic anhydrase from the soil bacterium Enterobacter sp. B13

A recombinant carbonic anhydrase (CA, EC 4.2.1.1) from the soil-dwelling bacterium Enterobacter sp. B13 was cloned and purified by Co²⁺ affinity chromatography. Bioinformatic analysis showed that the new enzyme (denominated here B13-CA) belongs to the β-class CAs and to possess 95% homology with the ortholog enzyme from Escherichia coli encoded by the can gene, whereas its sequence homology with the other such enzyme from E. coli (encoded by the cynT gene) was of 33%. B13-CA was characterized kinetically as a catalyst for carbon dioxide hydration to bicarbonate and protons. The enzyme shows a significant catalytic activity, with the following kinetic parameters at 20?°C and pH of 8.3: k_cat of 4.8?×?10⁵?s^?1 and k_cat/K_m of 5.6?×?10⁷ M^?1?×?s^?1. This activity was potently inhibited by acetazolamide which showed a K_I of 78.9?nM. Although only this compound was investigated for the moment as B13-CA inhibitor, further studies may reveal new classes of inhibitors/activators of this enzyme which may show biomedical or environmental applications, considering the posssible role of this enzyme in CaCO₃ biomineralization processes.     

Identification and characterization of a carboxysomal γ-carbonic anhydrase from the cyanobacterium Nostoc sp. PCC 7120

Carboxysomes are proteinaceous microcompartments that encapsulate carbonic anhydrase (CA) and ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco); carboxysomes, therefore, catalyze reversible HCO₃ ^? dehydration and the subsequent fixation of CO₂. The N- and C-terminal domains of the β-carboxysome scaffold protein CcmM participate in a network of protein–protein interactions that are essential for carboxysome biogenesis, organization, and function. The N-terminal domain of CcmM in the thermophile Thermosynechococcus elongatus BP-1 is also a catalytically active, redox regulated γ-CA. To experimentally determine if CcmM from a mesophilic cyanobacterium is active, we cloned, expressed and purified recombinant, full-length CcmM from Nostoc sp. PCC 7120 as well as the N-terminal 209 amino acid γ-CA-like domain. Both recombinant proteins displayed ethoxyzolamide-sensitive CA activity in mass spectrometric assays, as did the carboxysome-enriched TP fraction. NstCcmM209 was characterized as a moderately active and efficient γ-CA with a k _cat of 2.0 × 10⁴ s^?1 and k _cat/K _m of 4.1 × 10⁶ M^?1 s^?1 at 25 °C and pH 8, a pH optimum between 8 and 9.5 and a temperature optimum spanning 25–35 °C. NstCcmM209 also catalyzed the hydrolysis of the CO₂ analog carbonyl sulfide. Circular dichroism and intrinsic tryptophan fluorescence analysis demonstrated that NstCcmM209 was progressively and irreversibly denatured above 50 °C. NstCcmM209 activity was inhibited by the reducing agent tris(hydroxymethyl)phosphine, an effect that was fully reversed by a molar excess of diamide, a thiol oxidizing agent, consistent with oxidative activation being a universal regulatory mechanism of CcmM orthologs. Immunogold electron microscopy and Western blot analysis of TP pellets indicated that Rubisco and CcmM co-localize and are concentrated in Nostoc sp. PCC 7120 carboxysomes.     

In vitro and in vivo analyses of the role of the carboxysomal β-type carbonic anhydrase of the cyanobacterium Synechococcus elongatus in carboxylation of ribulose-1,5-bisphosphate

The carboxylase activities of crude carboxysome preparations obtained from the wild-type Synechococcus elongatus strain PCC 7942 strain and the mutant defective in the carboxysomal carbonic anhydrase (CA) were compared. The carboxylation reaction required high concentrations of bicarbonate and was not even saturated at 50 mM bicarbonate. With the initial concentrations of 50 mM and 25 mM for bicarbonate and ribulose-1,5-bisphosphate (RuBP), respectively, the initial rate of RuBP carboxylation by the mutant carboxysome (0.22 μmol mg^?1 protein min^?1) was only 30 % of that observed for the wild-type carboxysomes (0.71 μmol mg^?1 protein min^?1), indicating the importance of the presence of CA in efficient catalysis by ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). While the mutant defective in the ccmLMNO genes, which lacks the carboxysome structure, could grow under aeration with 2 % (v/v) CO₂ in air, the mutant defective in ccaA as well as ccmLMNO required 5 % (v/v) CO₂ for growth, indicating that the cytoplasmically localized CcaA helped utilization of CO₂ by the cytoplasmically localized Rubisco by counteracting the action of the CO₂ hydration mechanism. The results predict that overexpression of Rubisco would hardly enhance CO₂ fixation by the cyanobacterium at CO₂ levels lower than 5 %, unless Rubisco is properly organized into carboxysomes.     

Sulfonamide inhibition profile of the γ-carbonic anhydrase identified in the genome of the pathogenic bacterium Burkholderia pseudomallei the etiological agent responsible of melioidosis

A new γ-carbonic anhydrase (CA, EC 4.1.1.1) was cloned and characterized kinetically in the genome of the bacterial pathogen Burkholderia pseudomallei, the etiological agent of melioidosis, an endemic disease of tropical and sub-tropical regions of the world. The catalytic activity of this new enzyme, BpsCAγ, is significant with a k_cat of 5.3 × 10⁵ s^?1 and k_cat/K_m of 2.5 × 10⁷ M^?1 × s^?1 for the physiologic CO₂ hydration reaction. The inhibition constant value for this enzyme for 39 sulfonamide inhibitors was obtained. Acetazolamide, benzolamide and metanilamide were the most effective (K_Is of 149–653 nM) inhibitors of BpsCAγ activity, whereas other sulfonamides/sulfamates such as ethoxzolamide, topiramate, sulpiride, indisulam, sulthiame and saccharin were active in the micromolar range (K_Is of 1.27–9.56 μM). As Burkholderia pseudomallei is resistant to many classical antibiotics, identifying compounds that interfere with crucial enzymes in the B. pseudomallei life cycle may lead to antibiotics with novel mechanisms of action.     

Highly thermostable carbonic anhydrase from Persephonella marina EX-H1: Its expression and characterization for CO2-sequestration applications

The codon-optimized carbonic anhydrase gene of Persephonella marina EX-H1 (PMCA) was expressed and characterized. The gene with the signal peptide removed, PMCA(sp−), resulted in the production of approximately five times more purified protein than from the intact gene PMCA using an Escherichia coli expression system. PMCA(sp−) is formed as homo-dimer complex. PMCA(sp−) has a wide pH tolerance (optimum pH 7.5) and a high thermostability even at 100 °C (88 min of thermal deactivation half-life). The melting temperature for PMCA(sp−) was 84.5 °C. The apparent k_cat and K_m values for CO₂ hydration were 3.2 × 10⁵ s⁻¹ and 10.8 mM. The activity of the PMCA(sp−) enzyme was enhanced by Zn²⁺, Co²⁺, and Mg²⁺, but was strongly inhibited by Cu²⁺, Fe³⁺, Al³⁺, Pb²⁺, Ag⁺, and Hg²⁺. PMCA(sp−) readily catalyzed the hydration of CO₂, precipitating CaCO₃ as calcite in the presence of Ca²⁺.     

A one-step procedure for immobilising the thermostable carbonic anhydrase (SspCA) on the surface membrane of Escherichia coli

The carbonic anhydrase superfamily (CA, EC 4.2.1.1) of metalloenzymes is present in all three domains of life (Eubacteria, Archaea, and Eukarya), being an interesting example of convergent/divergent evolution, with its seven families (α-, β-, γ-, δ-, ζ-, η-, and θ-CAs) described so far. CAs catalyse the simple, but physiologically crucial reaction of carbon dioxide hydration to bicarbonate and protons. Recently, our groups characterised the α-CA from the thermophilic bacterium, Sulfurihydrogenibium yellowstonense finding a very high catalytic activity for the CO₂ hydration reaction (k_cat?=?9.35?×?10⁵?s^?1 and k_cat/K_m?=?1.1?×?10⁸?M^?1?s^?1) which was maintained after heating the enzyme at 80?°C for 3?h. This highly thermostable SspCA was covalently immobilised within polyurethane foam and onto the surface of magnetic Fe₃O₄ nanoparticles. Here, we describe a one-step procedure for immobilising the thermostable SspCA directly on the surface membrane of Escherichia coli, using the INPN domain of Pseudomonas syringae. This strategy has clear advantages with respect to other methods, which require as the first step the production and the purification of the biocatalyst, and as the second step the immobilisation of the enzyme onto a specific support. Our results demonstrate that thermostable SspCA fused to the INPN domain of P. syringae ice nucleation protein (INP) was correctly expressed on the outer membrane of engineered E. coli cells, affording for an easy approach to design biotechnological applications for this highly effective thermostable catalyst.     

Cardiolipin modulates allosterically the nitrite reductase activity of horse heart cytochrome c

Upon cardiolipin (CL) liposomes binding, horse heart cytochrome c (cytc) changes its tertiary structure disrupting the heme-Fe-Met80 distal bond, reduces drastically the midpoint potential, binds CO and NO with high affinity, displays peroxidase activity, and facilitates peroxynitrite isomerization. Here, the effect of CL liposomes on the nitrite reductase activity of ferrous cytc (cytc-Fe(II)) is reported. In the absence of CL liposomes, hexa-coordinated cytc-Fe(II) displays a very low value of the apparent second-order rate constant for the NO₂ ^?-mediated conversion of cytc-Fe(II) to cytc-Fe(II)-NO (k _on = (7.3 ± 0.7) × 10^?2 M^?1 s^?1; at pH 7.4 and 20.0 °C). However, CL liposomes facilitate the NO₂ ^?-mediated nitrosylation of cytc-Fe(II) in a dose-dependent manner inducing the penta-coordination of the heme-Fe(II) atom. The value of k _on for the NO₂ ^?-mediated conversion of CL-cytc-Fe(II) to CL-cytc-Fe(II)-NO is 2.6 ± 0.3 M^?1 s^?1 (at pH 7.4 and 20.0 °C). Values of the apparent dissociation equilibrium constant for CL liposomes binding to cytc-Fe(II) are (2.2 ± 0.2) × 10^?6 M, (1.8 ± 0.2) × 10^?6 M, and (1.4 ± 0.2) × 10^?6 M at pH 6.5, 7.4, and 8.1, respectively, and 20.0 °C. These results suggest that the NO₂ ^?-mediated conversion of CL-cytc-Fe(II) to CL-cytc-Fe(II)-NO could play anti-apoptotic effects impairing lipid peroxidation and therefore the initiation of the cell death program by the release of pro-apoptotic factors (including cytc) in the cytoplasm.     

The nature of anion inhibition of human red cell carbonic anhydrases.

We studied anionic inhibition of the reaction CO₂ + OH^?? HCO₃^? catalyzed by human red cell carbonic anhydrase B (I) and C (II), using iodide and cyanate. In the forward reaction with respect to CO₂ as the substrate, inhibition was mixed but favoring noncompetitive; the back reaction, with HCO₃^? as the substrate, yielded strict competitive kinetics. Mean inhibition constants, K_I, in the pH range 7.2–7.5 are: iodide, 0.5 mm for enzyme B and 16 mm for C; cyanate, 0.8 μm for B and 20 μm for C. When OH^? was considered as the substrate for the forward reaction, cyanate and chloride behaved as competitive inhibitors. The true inhibition constant (K_I⁰) for cyanate (calculated for infinitely low OH^?) is 0.4 μm for enzyme B and 4 μm for C. Apart from the difference in anion affinity and some 10-fold higher activity of C > B, the isozymes showed similar patterns of inhibition. Data agree with generally proposed mechanisms describing the active site as ZnH₂O with pK_a of about 7.     

Analysis of phospholipase C (Bacillus cereus) action toward mixed micelles of phospholipid and surfactant.

The action of phospholipase C (Bacillus cereus) toward mixed micelles of phosphatidylcholine and the nonionic surfactant Triton X-100 is analyzed according to the “surfaceas-cofactor” kinetic scheme recently proposed for characterizing the action of lipolytic enzymes [Deems, R. A., Eaton, B. R., and Dennis, E. A. (1975) J. Biol. Chem.250, 9013–9020]. According to this scheme, the enzyme first associates with the surface or mixed micelles, where the dissociation constant is K_s^A. The enzyme, now part of the mixed micelle surface, then binds the substrate phospholipid molecule in its active site and this binding is related to the Michaelis constant, K_m^B. The surface, or mixed micelles in this scheme, behaves kinetically as a cofactor in that, under initial rate conditions, the surface properties of the mixed micelles are virtually unchanged after catalysis. For phospholipase C with egg phosphatidylcholine as substrate, it was found that at pH 6.4 (the pH optimum for the enzyme) and 40 °C, V is about 2 × 10³ μmol min^?1 (mg of protein)^?1. K_s^A is about 2 mm and K_m^B is 1 to 2 × 10^?10 mol cm^?2. The kinetic constants for phospholipase C are compared with those previously reported for phospholipase A₂ and the membrane-bound enzyme phosphatidylserine decarboxylase determined under similar conditions.