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PubMed2004

X-ray and NMR characterization of covalent complexes of trypsin, borate, and alcohols.

An understanding of the physiological and toxicological properties of borate and the utilization of boronic acids in drug development require a basic understanding of borate-enzyme chemistry. We report here the extension of our recent NMR studies indicating the formation of a ternary borate-alcohol-trypsin complex. Crystallographic and solution state NMR studies of porcine trypsin were performed in the presence of borate and either of three alcohols designed to bind to the S1 affinity subsite: 4-aminobutanol, guanidine-3-propanol, and 4-hydroxymethylbenzamidine. Quaternary complexes of trypsin, borate, S1-binding alcohol, and ethylene glycol (a cryoprotectant), as well as a ternary trypsin, borate, and ethylene glycol complex have been observed in the crystalline state. Borate forms ester bonds to Ser195, ethylene glycol (two bonds), and the S1-binding alcohol (if present). Spectra from (1)H and (11)B NMR studies confirm that these complexes also exist in solution and also provide evidence for the formation of ternary trypsin, borate, and S1-subsite alcohol complexes which are not observed in the crystals using our experimental protocols. Analysis of eight crystal structures indicates that formation of an active site borate complex is in all cases accompanied by a significant (approximately 4%) increase in the b-axis dimension of the unit cell. Presumably, our inability to observe the ternary complexes in the crystalline state arises from the lower stability of these complexes and consequent inability to overcome the constraints imposed by the lattice contacts. A mechanism for the coupling of the lattice contacts with the active site that involves a conformational rearrangement of Gln192 is suggested. The structures presented here represent the first crystallographic demonstration of covalent binding of an enzyme by borate.

Amino Alcohols↗
PubMed2005

New bis-, tris- and tetrakis(pyrazolyl)borate ligands with 3-pyridyl and 4-pyridyl substituents: synthesis and coordination chemistry.

The new ligands dihydrobis[3-(4-pyridyl)pyrazol-1-yl]borate [Bp(4py)]-, hydrotris[3-(4-pyridyl)pyrazol-1-yl]borate [Tp(4py)]-, tetrakis[3-(4-pyridyl)pyrazol-1-yl]borate [Tkp(4py)]-, dihydrobis[3-(3-pyridyl)pyrazol-1-yl]borate [Bp(3py)]-, hydrotris[3-(3-pyridyl)pyrazol-1-yl]borate [Tp(3py)]- and tetrakis[3-(3-pyridyl)pyrazol-1-yl]borate [Tkp(4py)]- are derivatives of the well known bis-, tris- and tetrakis-(pyrazolyl)borate cores, bearing 4-pyridyl or 3-pyridyl substituents attached to the pyrazolyl C3 positions. These pyridyl groups cannot chelate to the metal ions in the poly(pyrazolyl) cavity but are externally directed. Structural studies on a range of metal complexes show how, in many cases, coordination of these pendant pyridyl groups to the M(pyrazolyl)n core of an adjacent metal complex fragment results in formation of coordination oligomers or polymeric networks. [Tl(Bp(3py))], [Tl(Bp(4py))] and [Tl(Tp(4py))] form one-dimensional polymeric chains via coordination of one of their pendant pyridyl units to the Tl(I) centre of an adjacent complex fragment; in contrast, in [Tl(Tp(3py))] coordination of all three pendant pyridyl units to separate Tl(I) neighbours results in formation of a two-dimensional polymeric sheet. In [Tl(Tkp(3py))] and [Tl(Tkp(4py))] the Tl(I) is coordinated by two or three of the four pyrazolyl arms, respectively; bridging interactions of pendant 4-pyridyl groups with adjacent Tl(I) centres result in a two-dimensional sheet forming in each case. In Ag(Tkp(4py)) each Ag(I) ion is coordinated by two pyrazolyl rings, and two bridging pyridyl ligands from other complex units, resulting in a one-dimensional chain consisting of pairs of cross-linked zigzag chains. In contrast to these polymeric coordination networks, the structures of [Cu(Tp(4py))] and [(Tp(3py))Cd(CH3CO2)] are dimers, with a pendant pyridyl residue from the first metal centre attaching to a vacant coordination site on the second, and vice versa; these dimers are stabilised by pi-stacking interactions between sections of the two ligands. [Ni(Tp(3py))2] is monomeric, with an octahedral coordination geometry arising from two tris(pyrazolyl)borate chelates; the array of pendant 3-pyridyl groups is involved only in intramolecular hydrogen-bonding. [(Tp(4py))Re(CO)3] is also monomeric, with a facial arrangement of three pyrazolyl ligands and three carbonyls, with the pendant 4-pyridyl groups not further coordinated. [(Tp(2py))Re(CO)3], based on the related ligand hydrotris[3-(2-pyridyl)pyrazol-1-yl]borate, has a similar fac-(CO)3(pyrazolyl)3 coordination geometry.

Journal Article↗
PubMed2004

Enantioseparations of hydrobenzoin and structurally related compounds in capillary zone electrophoresis using heptakis(2,3-dihydroxy-6-O-sulfo)-beta-cyclodextrin as chiral selector and enantiomer migration reversal of hydrobenzoin with a dual cyclodextrin system in the presence of borate complexation.

We investigated the enantioseparations of racemic hydrobenzoin, together with benzoin and benzoin methyl ether, in capillary electrophoresis (CE) using the single-isomer heptakis(2,3-dihydroxy-6-O-sulfo)-beta-cyclodextrin (SI-S-beta-CD) as a chiral selector in the presence and absence of borate complexation and enantiomer migration reversal of hydrobenzoin with a dual CD system consisting of SI-S-beta-CD and beta-CD in the presence of borate complexation at pH 9.0 in a borate buffer. The enantioselectivity of hydrobenzoin increased remarkably with increasing SI-S-beta-CD concentration and the enantioseparation depended on CD complexation between hydrobenzoin-borate and SI-S-beta-CD. The (S,S)-enantiomer of hydrobenzoin-borate complexes interacted more strongly than the (R,R)-enantiomer with SI-S-beta-CD. The enantiomers of hydrobenzoin could be baseline-resolved in the presence of SI-S-beta-CD at a concentration as low as 0.1% w/v, whereas the three test analytes were simultaneously enantioseparated with addition of 0.3% w/v SI-S-beta-CD or at concentrations >2.0% w/v in a borate buffer and 0.5% w/v in a phosphate background electrolyte at pH 9.0. Compared with the results obtained previously using randomly sulfated beta-CD (MI-S-beta-CD) in a borate buffer, enantioseparation of these three benzoin compounds is more advantageously aided by SI-S-beta-CD as the chiral selector. The enantioselectivity of hydrobenzoin depended greatly on the degree of substitution of sulfated beta-CD. Moreover, binding constants of the enantiomers of benzoin compounds to SI-S-beta-CD and those of hydrobenzoin-borate complexes to SI-S-beta-CD were evaluated for a better understanding of the role of CD complexation in the enantioseparation and chiral recognition. Enantiomer migration reversal of hydrobenzoin could be observed by varying the concentration of beta-CD, while keeping SI-S-beta-CD at a relatively low concentration. SI-S-beta-CD and beta-CD showed the same chiral recognition pattern but they exhibited opposite effects on the mobility of the enantiomers.

Benzoin↗
PubMed2004

Borate-nucleotide complex formation depends on charge and phosphorylation state.

Flow injection analysis with electrospray ionization mass spectrometry was used to investigate borate-nucleotide complex formation. Solutions containing 100 microM nucleotide and 500 microM boric acid in water-acetonitrile-triethylamine (50:50:0.2, v/v/v; pH 10.3) showed that borate complexation with nicotinamide nucleotides was significantly influenced by the charge on the nicotinamide group and the number of phosphate groups on the adenine ribose. Borate binding decreased in the order of NAD(+), NADH, NADP(+) and NADPH. To investigate the relationship between complex formation and phosphorylation, association constants (K(A)) of borate-adenine (AMP, ADP, ATP), -guanine (GMP, GDP, GTP), -cytidine (CMP, CDP, CTP) and -uridine (UMP, UDP, UTP) complexes were compared. The results showed that the number of nucleotide phosphate groups was inversely proportional to the relative abundance of the borate complexes, with the K(A) of borate-nucleotide complex decreasing in the order mono-, di- and tri-phosphates (AMP approximately GMP approximately CMP approximately UMP > ADP approximately GDP approximately CDP approximately UDP > GTP > ATP approximately CTP approximately UTP). At pH 7.4, using ammonium bicarbonate buffer, only borate-NAD(+) complex was observed. This indicates that the borate-NAD(+) complex may be the most physiologically relevant of those studied.

Borates↗
PubMed2004

Comparative studies on the enantioseparation of hydrobenzoin and structurally related compounds by capillary zone electrophoresis with sulfated beta-cyclodextrin as the chiral selector in the presence and absence of borate complexation.

Comparative studies on the enantioseparations of racemic hydrobenzoin, together with benzoin and benzoin methyl ether, in capillary zone electrophoresis using sulfated beta-cyclodextrin (S-beta-CD) as a chiral selector in the presence and absence of borate complexation were investigated. The influences of S-beta-CD concentration on the enantioseparation of benzoins in a borate buffer and a phosphate background electrolyte and the influences of the concentration and the pH of borate buffer containing S-beta-CD on the enantioseparation of hydrobenzoin were examined. The results indicate that, depending on the degree of strong borate complexation and comparatively weak CD complexation, the selectivity of the enantiomers of hydrobenzoin can be greatly reduced in a buffer system containing borate ions. Enantioseparation of hydrobenzoin is mainly governed by the interaction between hydrobenzoin-borate complexes and S-beta-CD in a borate buffer, whereas enantioseparation of benzoins is primarily determined by CD complexation in a phosphate background electrolyte. Effective enantioseparations of benzoins were simultaneously achieved with addition of S-beta-CD at a concentration greater than 3.0% (w/v) in a borate buffer and at a concentration greater than 2.5% (w/v) in a phosphate background electrolyte at pH 9.0.

Benzoin↗
PubMed1991

Borate interference in surface-enhanced Raman spectroscopy of amines.

Interference from borate is observed in surface-enhanced Raman (SER) spectra of lysine and propylamine obtained with borohydride-reduced silver colloids. Borate bands are also observed in the spectra of other basic analytes, as well as when certain variations are made in the silver colloid preparation. The relative intensities of the analyte and borate bands depend on the pH of the colloid, the extent of oxidation of the colloid surface, and the relative adsorptivities of the analyte and borate. Benzylamine adsorbs more readily than propylamine and also competes more effectively with borate for adsorption sites. On the other hand, borate virtually excludes lysine from the surface when the solution pH is greater than or equal to 8. The formation of silver oxide in basified colloids may facilitate borate adsorption. For some basic analytes, eliminating the adsorption of borate ion and the resulting spectral interference may require using alternative SERS substrates.

Amines↗
PubMed2004

NaBC1 is a ubiquitous electrogenic Na+ -coupled borate transporter essential for cellular boron homeostasis and cell growth and proliferation.

Boron is a vital micronutrient in plants and may be essential for animal growth and development. Whereas the role of boron in the life cycle of plants is well documented, nothing is known about boron homeostasis and function in animal cells. NaBC1, the mammalian homolog of AtBor1, is a borate transporter. In the absence of borate, NaBC1 conducts Na(+) and OH(-) (H(+)), while in the presence of borate, NaBC1 functions as an electrogenic, voltage-regulated, Na(+)-coupled B(OH)(4)(-) transporter. At low concentrations, borate activated the MAPK pathway to stimulate cell growth and proliferation, and at high concentrations, it was toxic. Accordingly, overexpression of NaBC1 shifted both effects of borate to the left, whereas knockdown of NaBC1 halted cell growth and proliferation. These findings may reveal a previously unrecognized role for NaBC1 in borate homeostasis and open the way to better understanding of the many presumed physiological roles of borate in animals.

Animals↗
PubMed2002

Potential binding of borate ions to mono- and oligonucleotides: a capillary electrophoresis investigation.

The potential binding of borate to oligonucleotides and DNA fragments is here investigated. In case of free nucleotides, such as AMP, there appears to be a weak binding, although no free versus complexed species could ever be separated under any experimental condition. The binding was suggested by the strong peak asymmetry and by the fact that, at progressively lower borate molarities in the background electrolyte, the peak shape suddenly switched from fronting to tailing. This indicated, as also confirmed by theoretical simulations, that the AMP-borate complex was the slow, not the fast moving species. On the contrary, in the case of free adenosine, strong binding ensued, since in Tris-acetate buffer this compound was only eluted with the electroendoosmotic flux, being neutral, whereas in Tris-borate it had a much higher mobility, comparable to, although lower than, that of AMP. When running oligonucleotides, at standard borate molarities (ca. 45 mM), and under strict iso-ionic strength conditions, no binding to borate could be demonstrated, since the free mobility of a 24-mer DNA was identical in TA and TB buffers. However, at very high borate molarities (200 mM) and high pH values (pH 8.92), some binding to oligonucleotides could occur, since in these latter conditions the mobility of a 24-mer was seen to be ca. 20% lower than at pH 7.69, a pH value that should discourage any complex formation.

Borates↗
PubMed1999

Low-level calibration study for a new ion chromatographic column to determine borate in deionized water.

In the semiconductor industry, there is interest in determining borate at sub-ppb levels in ultrapure water, since borate is an early breakthrough ion from ion-exchange resin beds. Although dissolved silica is the most common species currently used to monitor the breakdown of the deionization systems, it is thought that borate probably breaks through earlier than silicate. To be of use as an early-warning indicator, borate must be determined at ppt levels. This paper discusses benchtop results with several new column products designed to deliver low-ppt detection limits for boron as borate. The system uses a prototype borate-specific concentrator column that is coupled to an ion-exclusion separator and suppressed-conductivity detection. The acidic eluent, containing mannitol, quantitatively elutes the borate from the concentrator. The analytical separation is performed using a specially designed ion-exclusion column. Data presented are from two multilevel calibration studies. Included is a discussion of detection-limit calculations and recommended formats for reporting results.

Benchmarking↗
PubMed1976

Borate inhibition of yeast alcohol dehydrogenase.

Yeast alcohol dehydrogenase is inhibited competitively by borate with respect to NAD+. An unusual mechanism of competitive inhibition prevails: the competition for the substrate NAD+ by borate and enzyme. The following evidence supports this conclusion. (1) Much greater inhibition is observed with respect to NAD+ as compared with NADH as substrates. (2) Borate decreases the equilibrium constant of the overall reaction in the direction of ethanol oxidation, therefore, borate enters directly into the overall reaction rather than merely decreases the effectiveness of the catalyst. (3) The Ki values for unrelated enzyme reactions are identical for NAD+. (4) Stopped-flow experiments show burst kinetics only when NAD+ and borate are not premixed. (5) The Ki value is identical with the inverse of the borate-NAD+ complexation constant. (6) The pH dependence of the inhibitor demonstrates that only the B(OH)4-species is inhibiting. These results are consistent with the preferable binding of borate to NAD+ as compared with NADH. These two binding constants were found to be equal to 2000 +/- 60 and 130 +/- 8 M-1, respectively. In contrast to the liver enzyme, the yeast enzyme does not show pre-steady-state burst reactions in the reduction of NAD+. This would indicate that the interconversion of ternary complexes is at least partially rate limiting for the yeast enzyme.

Alcohol Oxidoreductases↗
PubMedDate not supplied

Issues concerning the measurement of borate in occupational environments.

Borates are susceptible to weight change due to uptake or loss of water and this hydration instability can lead to gravimetric and interpretation errors in occupational hygiene field sampling of dust. The hydration stability for inhalable borate dust particles (mean diameter 7-22 microns) was characterized over a range of ambient temperature and relative humidity conditions simulating field sampling. Borax 10 mol (Na2O.2B2O3.10H2O), a fully hydrated borate, has a relatively high vapor pressure to water that led to rapid dehydration with significant weight change. Low hydrate borates, Neobor borax 5 mol (Na2O.2B2O3.5H2O), anhydrous boric acid (B2O3), and anhydrous borax (Na2O.2B2O3) were found to hydrate rapidly with an increase in weight. In contrast, boric acid (B[OH]3) and borax 5 mol were found to be stable to dehydration under all conditions. Boron can be measured with high analytical accuracy, but because the specific borate species or borate compounds collected in a 37-mm dust sampler cannot be accurately identified, it is argued that occupational exposure values should be revised to reflect exposure to boron and exposure values for these borates should be the same based on equivalent boron content.

Borates↗
PubMed1999

Amine borate catabolism by bacteria isolated from contaminated metal-working fluids

Four bacterial strains (tentatively identified as strains of Aeromonas, Pseudomonas, Flavobacterium and Bacillus) isolated from contaminated metal-working fluids were assayed for the capacity to utilize the borate derivatives of monoethanolamine (MEA), diethanolamine (DEA) and triethanolamine (TEA). Two of these strains, isolates AV1 (Flavobacterium) and CL1 (Bacillus) were capable of growth on each of the borate esters with cell yields of 0.6 gl - 1 for AV1 cultured on DEA- and TEA-borate, 0.3-0.4 gl - 1 for CL1 cultured on DEA- and TEA-borate and approximately 1.4 gl - 1 for AV1 and CL1 cultured on MEA-borate. In the case of strain CL1, growth yields on TEA- or DEA-borate as substrates were doubled by the addition of potassium ions. Lower ethanolamines, glycolaldehyde, acetaldehyde and ammonia were identified as breakdown products. The enzymes produced during growth upon the alkanolamine borates were shown to possess similar properties to those seen for cells cultured upon alkanolamine hydrochlorides.

Journal Article↗
PubMed2004

Tris-borate is a poor counterion for RNA: a cautionary tale for RNA folding studies.

Native polyacrylamide gel electrophoresis is a powerful approach for visualizing RNA folding states and folding intermediates. Tris-borate has a high-buffering capacity and is therefore widely used in electrophoresis-based investigations of RNA structure and folding. However, the effectiveness of Tris-borate as a counterion for RNA has not been systematically investigated. In a recirculated Hepes/KCl buffer, the catalytic core of the bI5 group I intron RNA undergoes a conformational collapse characterized by a bulk transition midpoint, or Mg1/2, of approximately 3 mM, consistent with extensive independent biochemical experiments. In contrast, in Tris-borate, RNA collapse has a much smaller apparent Mg1/2, equal to 0.1 mM, because in this buffer the RNA undergoes a different, large amplitude, folding transition at low Mg2+ concentrations. Analysis of structural neighbors using a short-lived, RNA-tethered, photocrosslinker indicates that the global RNA structure eventually converges in the two buffer systems, as the divalent ion concentration approaches approximately 1 mM Mg2+. The weak capacity of Tris-borate to stabilize RNA folding may reflect relatively unfavorable interactions between the bulky Tris-borate ion and RNA or partial coordination of RNA functional groups by borate. Under some conditions, Tris-borate is a poor counterion for RNA and its use merits careful evaluation in RNA folding studies.

Base Sequence↗
PubMed1979

Borate Exchanges of Lemna minor L. as Studied with the Help of the Enriched Stable Isotopes and of a (n,alpha) Nuclear Reaction.

Despite the lack of a convenient radioisotope of boron, it is possible to measure unidirectional fluxes of borate between cellular systems and their external medium. It was accomplished by using the two purified stable isotopes ((10)B and (11)B), with (10)B specifically detected by a (n,alpha) nuclear reaction. The method was applied to compartmental analysis of borate with intact plants of Lemna minor L. Four compartments were suggested. Three of them apparently correspond to the three classical ones: free space (including easily dissociable borate monoesters), cytoplasm, and vacuole. The fourth one was interpreted as corresponding to very stable borate diesters in the cell walls. The method allows the determination of the borate capacities of the various compartments and of the borate unidirectional fluxes between the different compartments, at borate flux equilibrium. Other physicochemical data (mono and diester mass action constants, turn over numbers) were evaluated. The results are consistent with what is known of pure substances.

Journal Article↗
PubMed1993

Separation of N-acetylglucosamine and N-acetylmannosamine by chromatography on Sephadex in borate buffer.

Several procedures have been used previously for the separation of N-acetylglucosamine and N-acetylmannosamine, which are based on the difference in strength of the borate complexes of the two N-acetylhexosamines and include paper chromatography on borate-treated paper, paper electrophoresis in borate buffer, and anion-exchange chromatography of the borate complexes. In the present study, we have observed that the two sugars, despite their identical size in noncomplexed form, may also be separated by gel chromatography in borate buffer. Nearly complete resolution was obtained by chromatography on a column (1.5 x 117 cm) of Sephadex G-15, which was eluted at room temperature with 0.27 M sodium borate, pH 7.8 (prepared from H3BO3 by addition of NaOH), at a flow rate of 10 ml/h. This procedure complements existing methods for the separation of N-acetylmannosamine and N-acetylglucosamine and has the advantage that it can be carried out on a relatively large preparative scale.

Acetylglucosamine↗
PubMed1993

Borate ion-assisted stabilization of beta-galactosidase from Aspergillus oryzae by polyhydroxy compounds in water-miscible organic solvents.

The stability of beta-galactosidase from Aspergillus oryzae in water-miscible organic solvents in different buffers at various pH values ranging from 4.6 to 8.0 was studied. The stability of the enzyme in all six organic solvents studied was dependent on pH and on the type of buffer ions present. At a given pH, destabilization by organic solvents was highest in sodium borate buffer. The destabilization of beta-galactosidase by these solvents could be reversed by addition of sugars or polyhydroxy compounds exclusively in sodium borate, suggesting a role of borate ions in stabilization. A similar effect of addition of mannitol was observed on deactivation of beta-galactosidase by N, N-dimethylformamide (DMF). Exclusively in sodium borate, at pH 8.0, the addition of mannitol (0.02 M) not only prevented the deactivation by DMF (8%, v/v) but increased the enzyme activity to the level at its optimum pH. Since beta-galactosidase from Aspergillus oryzae is a glycoprotein, complexation of the borate ions to the carbohydrate part may result in change in protein conformation, which, without leading to denaturation or inactivation of the enzyme, may facilitate interaction of the organic solvents with the enzyme leading to its denaturation. Such a denaturation is probably prevented by addition of polyhydroxy compounds, which appear to compete favorably with the carbohydrate moiety of the protein in complexing with borate ions. This should result in the enzyme regaining its native conformation.

Aspergillus oryzae↗
PubMed2004

Enantioseparation of benzoins and enantiomer migration reversal of hydrobenzoin in capillary zone electrophoresis with dual cyclodextrin systems and borate complexation.

Enantioseparations of racemic hydrobenzoin and structurally related compounds, including benzoin and benzoin methyl ether, in capillary zone electrophoresis (CZE) with dual cyclodextrin (CD) systems consisting of S-beta-CD (mixed isomers) and a neutral CD, including beta-CD and hydroxypropyl-beta-CD (HP-beta-CD), as chiral selectors in the presence of borate complexation at pH 9.0 were investigated. Effective enantioseparations of hydrobenzoin were achieved with addition of dual CD systems and also with neutral CDs in a borate buffer. The enantioseparation and migration behavior of hydrobenzoin in such an electrophoretic system are primarily governed by the interaction of the borate complex of hydrobenzoin with beta-CDs. The CD complexations of both hydrobenzoin and the borate complexes of hydrobenzoin with beta-CDs increase in the order S-beta-CD < HP-beta-CD < beta-CD. As a result, enantioseparations of hydrobenzoin with the use of dual CD systems consisting of S-beta-CD/beta-CD and S-beta-CD/HP-beta-CD as chiral selectors are more advantageous than that with the use of S-beta-CD alone. With these dual CD systems in the presence of borate complexation, the enantiomer migration reversal was observed for hydrobenzoin. The interactions of hydrobenzoin with neutral CDs and with S-beta-CD exhibit the same chiral recognition pattern, but opposite effect on the mobility of the enantiomers. The (S,S)-enantiomer of hydrobenzoin was found to interact more strongly than the (R,R)-enantiomer with neutral CDs. For comparison, enantioseparation of hydrobenzoin, together with benzoin and benzoin methyl ether, with dual CD systems in a phosphate background electrolyte at pH 9.0 was also examined. The migration order and enantioselectivity of these three benzoins depend on the degree of CD complexations between benzoins and both S-beta-CD and neutral CD in a phosphate background electrolyte. In addition, effective enantioseparations of hydrobenzoin were also achievable with addition of either beta-CD at concentrations greater than 1.0 mM or HP-beta-CD at concentrations exceeding 2.0 mM in a borate buffer at pH 9.0.

Benzoin↗
PubMedDate not supplied

Manganese-dependent inhibition of human liver arginase by borate.

Full activation of human liver arginase (EC 3.5.3.1), by incubation with 5 mM Mn2+ for 10 min at 60 degrees C, resulted in increased Vmax and a higher sensitivity of the enzyme to borate inhibition, with no change in the K(m) for arginine. Borate behaved as an S-hyperbolic I-hyperbolic non-competitive inhibitor and had no effect on the interaction of the enzyme with the competitive inhibitors L-ornithine (Ki = 2 +/- 0.5 mM), L-lysine (Ki = 2.5 +/- 0.4 mM), and guanidinium chloride (Ki = 100 +/- 10 mM). The pH dependence of the inhibition was consistent with tetrahedral B(OH)4- being the inhibitor, rather than trigonal B(OH)3. We suggest that arginase activity is associated with a tightly bound Mn2+ whose catalytic action may be stimulated by addition of a more loosely bound Mn2+, to generate a fully activated enzyme form. The Mn2+ dependence and partial character of borate inhibition are explained by assuming that borate binds in close proximity to the loosely bound Mn2+ and interferes with its stimulatory action. Although borate protects against inactivation of the enzyme by diethyl pyrocarbonate (DEPC), the DEPC-sensitive residue is not considered as a ligand for borate binding, since chemically modified species, which retain about 10% of enzymatic activity, were also sensitive to the inhibitor.

Arginase↗