pH kinetic studies of the N-demethylation of N,N-dimethylaniline catalyzed by chloroperoxidase

The effect of pH on the kinetic parameters for the chloroperoxidase-catalyzed N-demethylation of N,N-dimethylaniline supported by ethyl hydroperoxide was investigated from pH 3.0 to 7.0. Chloroperoxidase was found to be stable throughout the pH range studied. Initial rate conditions were determined throughout the pH range. The V_max for the demethylation reaction exhibited a pH optimum at approximately 4.5. The K_m for N,N-dimethylaniline increased with decreasing pH, while the K_m for ethyl hydroperoxide varied in a manner paralleling V_max. Comparison of the $\text{V}{}_{\mathrm{<mtext>max</mtext>}}\text{K}{}_{\mathrm{m}}$ values for N,N-dimethylaniline and ethyl hydroperoxide indicated that the interaction of N,N-dimethylaniline with chloroperoxidase compound I was rate-limiting below pH 4.5, while compound I formation was rate-limiting above pH 4.5. The log of the $\text{V}{}_{\mathrm{<mtext>max</mtext>}}\text{K}{}_{\mathrm{m}}$ for ethyl hydroperoxide was independent of pH, indicating that chloroperoxidase compound I formation is not affected by ionizations in this pH range. The plot of the log of the $\text{V}{}_{\mathrm{<mtext>max</mtext>}}\text{K}{}_{\mathrm{m}}$ for N,N-dimethylaniline versus pH indicated an ionization on compound I with a pK of approximately 6.8. The plot of the log of the V_max versus pH indicated an ionization on the compound I-N,N-dimethylaniline complex, with a pK of approximately 3.1. The results show that chloroperoxidase can demethylate both the protonated and neutral forms of N,N-dimethylaniline (pK approximately 5.0), suggesting that hydrophobic binding of the arylamine substrate is more important in catalysis than ionic bonding of the amine moiety. For optimal catalysis, a residue in the chloroperoxidase compound I-N,N-dimethylaniline complex with a pK of approximately 3.1 must be deprotonated, while a residue in compound I with a pK of approximately 6.8 must be protonated.     

Purification and Characterization of an Aminopeptidase from Lactococcus lactis subsp. cremoris Wg2

An aminopeptidase was purified to homogeneity from a crude cell extract of Lactococcus lactis subsp. cremoris Wg2 by a procedure that included diethyl-aminoethane-Sephacel chromatography, phenyl-Sepharose chromatography, gel filtration, and high-performance liquid chromatography over an anion-exchange column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme showed a single protein band with a molecular weight of 95,000. The aminopeptidase was capable of degrading several peptides by hydrolysis of the N-terminal amino acid. The peptidase had no endopeptidase or carboxypeptidase activity. The aminopeptidase activity was optimal at pH 7 and 40°C. The enzyme was completely inactivated by the p-chloromecuribenzoate mersalyl, chelating agents, and the divalent cations Cu²⁺ and Cd²⁺. The activity that was lost by treatment with the sulfhydryl-blocking reagents was restored with dithiothreitol or β-mercapto-ethanol, while Zn²⁺ or Co²⁺ restored the activity of the 1,10-phenantroline-treated enzyme. Kinetic studies indicated that the enzyme has a relatively low affinity for lysyl-p-nitroanilide (K_m, 0.55 mM) but that it can hydrolyze this substrate at a high rate (V_max, 30 μmol/min per mg of protein).     

A glucose-tolerant β-glucosidase from Prunus domestica seeds: Purification and characterization

A glucose-tolerant β-glucosidase was purified to homogeneity from prune (Prunus domestica) seeds by successive ammonium sulfate precipitation, hydrophobic interaction chromatography and anion-exchange chromatography. The molecular mass of the enzyme was estimated to be 61 kDa by SDS-PAGE and 54 kDa by gel permeation chromatography. The enzyme has a pI of 5.0 by isoelectric focusing and an optimum activity at pH 5.5 and 55 °C. It is stable at temperatures up to 45 °C and in a broad pH range. Its activity was completely inhibited by 5 mM of Ag⁺ and Hg²⁺. The enzyme hydrolyzed both p-nitrophenyl β-d-glucopyranoside with a K_m of 3.09 mM and a V_max of 122.1 μmol/min mg and p-nitrophenyl β-d-fucopyranoside with a K_m of 1.65 mM and a V_max of 217.6 μmol/min mg, while cellobiose was not a substrate. Glucono-δ-lactone and glucose competitively inhibited the enzyme with K_i values of 0.033 and 468 mM, respectively.     

Purification and characterization of a lysozome from wheat germ

Several proteins of wheat germ were able to lyse Micrococcus luteus cells. One lysozyme, named W1A, was purified by ammonium sulfate fractionation, ion-exchange chromatography, gel filtration and preparative polyacrylamide gel electrophoresis (PAGE) under native conditions. The enzyme had a molecular weight of 25 400 as determined by sodium dodecyl sulfate (SDS)-PAGE. The reducing groups released from the lysis of Micrococcus cell walls by W1A lysozyme were $\text{N-}\text{acetylmuramic}$ acid residues as for hen egg white lysozyme (HEWL). Chitin substrates were hydrolyzed to some extent by this enzyme. With Micrococcus cells as substrate, the pH optimum for W1A lysozyme was 6.0 at an optimal ionic strength of 0.05. Under these conditions, the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value was 166 mg/l with purified Micrococcus cell walls and the V_max value was 0.56 A₅₄₀ unit/min at 22°C. W1A lysozyme exhibited the highest lytic activity at 60°C whereas the enzyme was inactive above 90°C. W1A lysozyme was strongly inhibited by poly-l-lysine and glycol chitosan. This is the first report of the presence of multiple electrophoretic forms of plant lysozyme activity as determined by native PAGE.     

Glutathione peroxidase activity of glutathione-S-transferases purified from rat liver

Gel filtration chromatography demonstrated the presence of two peaks of glutathione peroxidase activity assayed with cumene hydroperoxide in the soluble fraction of rat liver, brain, kidney, and testis. The peak with an approximate molecular weight of 45,000 (GSH-Px II) was purified from rat liver labeled $\text{in vivo}$ with Na₂⁷⁵SeO₃. Chromatography on DEAE-cellulose, Sephadex G-150, DEAE-cellulose, and CM-cellulose resulted in the co-purification of glutathione-S-transferase activity measured with 1-chloro-2,4-dinitrobenzene and glutathione peroxidase activity assayed with cumene hydroperoxide, and in the removal of all detectable ⁷⁵Se. Studies on GSH-Px II indicated that the apparent K_m for both cumene and t-butyl hydroperoxides was considerably higher than that for purified seleno-glutathione peroxidase. The V_max estimated with cumene hydroperoxide was only $\text{1}\text{300}$ of that determined for the selenoenzyme at pH 7.5 and with 1 mM GSH.     

D-Fructose 2,6-bisphosphate: a naturally occurring activator for inorganic pyrophosphate:D-fructose-6-phosphate 1-phosphotransferase in plants

Inorganic pyrophosphate:D-fructose-6-phosphate 1-phosphotransferase from mung beans ($\text{Phaseolus}\text{aureus}\text{Roxb.}$) was activated markedly by D-fructose 2,6-bisphosphate, with a K_A of about 50 nM. The enzyme exhibited hyperbolic kinetics both in the absence and presence of the activator. D-Fructose 2,6-bisphosphate (1 μM) decreased the K_m for D-fructose 6-phosphate 67-fold (from 20 mM to 0.3 mM) and increased the V_max 15-fold; these two effects combined to give a 500-fold activation at 0.3 mM D-fructose 6-phosphate. In contrast, ATP:D-fructose 6-phosphate 1-phosphotransferase from the same source was found not to be affected by D-fructose 2,6-bisphosphate.A natural activator for inorganic pyrophosphate:D-fructose 6-phosphate 1-phosphotransferase was isolated from mung-bean extracts and identified as D-fructose 2,6-bisphosphate.     

Effects of divalent cations on the activity of ribulosebisphosphate carboxylase: interactions with pH and with D2O as solvent

Maximum velocity-pH profiles have indicated that one ionizing group on the quinary complex of ribulosebisphosphate carboxylase must be dissociated and possibly a second protonated for catalytic activity. The pK values for the soybean enzyme are 7.5 and 9.1, respectively. A third group, identified from $\text{V}\text{K}\text{-}\text{pH}$ profiles for carbon dioxide as the variable substrate and having a pK of 8.1 for the soybean enzyme, must be dissociated for the binding of carbon dioxide. Only the pK at 7.5 is changed when manganese is substituted for magnesium, whereas only the pK at 8.1 is altered when deuterium oxide is substituted for water as solvent. Small but different solvent isotope effects are observed in the maximum velocity with the two cations.     

Insulin and glucagon's reciprocal mediation of a dual regulation mechanism for pyruvate kinase

The addition of glucagon to hepatocytes in primary culture produced a rapid and sustained increase in the K_m ($\text{1.27 m}\text{M}$ phosphoenol pyruvate) of pyruvate kinase. The low K_m ($\text{0.4 m}\text{M}$) form of the enzyme was seen when cells were retreated with insulin, demonstrating a short-term regulation mechanism. Injections of insulin, glucagon or glucagon followed by insulin demonstrated that a similar mechanism occurs $\text{in}\text{vivo}$. Results from longer times after injection indicated that another mechanism occurs when altered activity was the result of changes in V_max and not K_m. Thus, a dual mechanism for regulation of pyruvate kinase occurs. A rapid responding system functions by modification of the enzyme, while a long-term system functions by altering the rate of synthesis, thus changing the amount of enzyme present.     

Effect of pH on the Kinetic Parameters of NADP-Malic Enzyme from a C(4)Flaveria (Asteraceae) Species

We have used the pH variation in the kinetic parameters with respect to malate of NADP-malic enzyme purified from the C₄ species, Flaveria trinervia, to compare the pK values of its functional groups with those for the pigeon liver NADP-malic enzyme (MI Schimerlik, WW Cleland [1977] Biochemistry 16: 576-583) and the plant NAD-malic enzyme (KO Willeford, RT Wedding [1987] Plant Physiol 84: 1084-1087). Like the other enzymes, the C₄ enzyme has a group with a pK of about 6.0 (6.6 for the C₄ enzyme), as indicated from plots of the log V_max/K_m (V_max = maximum rate of catalysis) versus pH, which must lose a proton for malate binding and subsequent catalysis. The optimum ionization for the C₄ enzyme-NADP-Mg²⁺ complex occurs at pH 7.1 to 7.5. From pH 7.5 to 8.4, the K_m increases, but V_max remains constant. The log V_max/K_m plot in this pH range indicates a group with a pK of about 7.7. The other malic enzymes exhibit a similar pK. Above pH 8.4, deprotonation leads to a marked increase in K_m and a decrease in V_max for the C₄ enzyme. As in the case of the animal enzyme, the log V_max/K_m plot for the C₄ enzyme appears to approach a slope of two. The curve suggests an average pK of 8.4 for the groups involved, while the animal enzyme exhibits an average pK of 9.0. The NAD-malic enzyme does not exhibit any pK values at these high pK values. We hypothesize that the putative groups with the high pK values may be at least partially responsible for the ability of the C₄ NADP-malic enzyme to maintain high activity at pH 8.0 in illuminated chloroplasts.     

Trichomonas vaginalis: NADH oxidase activity.

NADH oxidase activity was detected in the 105,000g supernatant (“soluble”) fraction of Trichomonas vaginalis and the enzyme was purified 50-fold by centrifugation, ammonium sulfate precipitation, Sephadex G-200, and DEAE-Sephadex A-25 chromatography. The ratio of oxygen uptake to NADH oxidation was approximately one-half. Addition of catalase did not affect the rate of oxygen uptake elicited by NADH. Since the purified fraction was free from interfering enzymes, the postulated reaction is as follows: $\text{NADH}\text{+}\text{H}{}^{\mathrm{+}}\text{+}\text{1}\text{2}\text{= NAD}{}^{\mathrm{+}}\text{+ H}{}_2\text{O}$. Among numerous substances tested, only NADH was a functional substrate, whereas NADPH was not oxidized. The purified enzyme had a V_max of 16.5 μmole of oxygen consumed/min/mg protein, and the apparent K_m for NADH was 7.4 μM. Substrate inhibition was observed at 3.7 mM NADH. The purified NADH oxidase was competitively inhibited by NAD⁺ as well as by NADP⁺ with 50% inhibition at 1 and 5 mM, respectively. The enzyme was also markedly inhibited by p-chloromercuribenzoate, hydrogen peroxide, and transient metal-chelators such as bathophenanthroline or o-phenanthroline. A flavoprotein antagonist, atebrin was slightly less inhibitory. Various quinones, flavin nucleotides and artificial dyes, except for p-benzoquinone, ferricyanide and cytochrome c, did not function in accepting electrons from NADH oxidase. These three compounds, however, were still poor electron acceptors in the enzymatic reaction suggesting that the trichomonad NADH oxidase has little diaphorase activity. All of these findings indicate that T. vaginalis has an unique NADH oxidizing enzyme in that H₂O seems to be the prdouct of oxygen reduction. This NADH oxidase appears important in the aerobic metabolism of this parasite.     

Breaks in arrhenius plots of reactions involving membrane-bound and solubilized sialyltransferases,due to temperature dependence of kinetic parameters

Temperature dependence of asialomucin-sialyltransferase ($\text{CMP}\text{-N-}\text{acetylneuraminate:}\text{D}\text{-galactosyl-glyco-protein}$) N-acetylneuraminyltransferase, EC 2.4.99.1) activity is investigated. Discontinuities in Arrhenius plots are observed, whether the enzyme is membrane-associated or solubilized. These discontinuities cannot be firmly correlated with the phase-transition temperatures of either endogenous or exogenous phospholipids. Arrhenius plots of the kinetic parameters also exhibit sharp discontinuities, so that it is concluded that a significant change in K_m and V_max values occurs with varying temperature. Our results suggest that the biphasic behavior of Arrhenius plots may be attributed to the temperature dependence of the kinetic parameters for both membrane-associated and solubilized sialyltransferase activities.     

Foundations of the use of an enzyme-kinetic analogy in cell-mediated cytotoxicity

A mathematical model of the ⁵¹Cr-release microcytotoxicity assay is utilized to find conditions under which the kinetics of this assay resemble the kinetics of a classical enzyme-substrate reaction. Assuming a steady-state approximation, that “bystander” effector cells do not bind markedly better than the cytotoxic effector cells, and that the programming of the target cells for lysis is irreversible, it is shown that the velocity of label release is v = v_maxT_T/(K_{$\text{1}\text{2}$}+T_T), where both V_max and K_{$\text{1}\text{2}$} are linear functions of the effector-cell population and T_T is the initial target-cell population. Moreover, the expressions for K_{$\text{1}\text{2}$} and V_max are expressed in terms of natural kinetic parameters of the process and attributes of the noncytotoxic bystanders.     

Purification and characterization of a secreted purple phosphatase from soybean suspension cultures

We purified and partially sequenced a purple (λ_max = 556 nanometers) acid phosphatase (APase; EC 3.1.3.2) secreted by soybean (Glycine max) suspension-culture cells. The enzyme is a metalloprotein with a Mn²⁺ cofactor. This APase appears to be a glycoprotein with a monomer subunit molecular weight of 58,000 and an active dimer molecular weight of approximately 130,000. The protein has an isoelectric point of about 5.0 and a broad pH optimum centered near 5.5. The purified enzyme, assayed with p-nitrophenyl phosphate as the substrate, has a specific activity of 512 units per milligram protein and a K_m of approximately 0.3 millimolar; phosphate is a competitive inhibitor with a K_i of 0.7 millimolar. This APase is similar to one found in soybean seed meal but dissimilar to that found in soybean seedlings.     

Physico-kinetic and functional features of a novel β-glucosidase isolated from milk thistle (Silybum marianum Gaertn.) flower petals

A highly abundant β-glucosidase from petals of Silybum marianum has been purified and characterized for its physico-kinetic properties. The 135 kDa enzyme was a homodimer with subunit molecular mass of 67.6 kDa. The characteristic catalytic properties of the enzyme included acidic pH optimum (5.5), meso-thermostability, and β-linked substrate specificity with preference for gluco-conjugate but a marked (>50 %) activity with D-fuco-conjugates and considerable (~16 %) activity towards D-galacto-conjugates. The enzyme showed high affinity for p-nitrophenyl glucoside (pNPG) with K_m and V_max values of 0.25 mM and 5.35 μkat.mg^?1 enzyme protein. Thus, the enzyme had a very high (292,000 M^?1.s^?1) catalytic efficiency (K_cat/K_m). Thermal catalytic optimum of enzyme was 40 °C with activation energy value 8.26 kCal.Mol^?1. The enzyme showed significant insensitivity to D-gluconic acid lactone inhibition (57 % at 5 mM) with an apparent K_i 3.8 mM. The transglucosylating ability of enzyme was noticed for glucosylation of geraniol and withaferin-A with pNPG as glucosyl donor but cellobiose did not serve as the glycosyl donor. Partial proteomics of the enzyme revealed two peptide fragment sequences, VTPSNEVH and KRSEESNF. These motifs showed significant matching/sequence conservation with some other glycohydrolases. The novelties of purified enzyme hold potential to expand a library of catalytically characteristic members of the hydrolase family from plants for use in biotransformation applications.     

The pH dependence of peptide hydrolysis by nitrocarboxypeptidase A

Hydrolysis of benzyloxycarbonyl-GlyGlyPhe by nitro(Tyr 248)carboxypeptidase A over the pH range 4.88–8.04 has been examined. The nitroenzyme retains appreciable activity near pH 6.5, and the limiting value of K_m is scarcely affected. The peptidase activity has a pH dependence characterized by the following parameters: pK_E1 of 6.37 ± 0.19 and pK_E2 of 6.60 ± 0.17 in $\text{k}{}_{\mathrm{<mtext>cat</mtext>}}\text{K}{}_{\mathrm{m}}$, and apparent pK of 5.59 ± 0.06 in K_cat. A spectroscopic pK of 6.75 ± 0.01, attributable to the nitro-Tyr 248 residue, has been determined. This correlates with the base-limb pK_E2 in the $\text{k}{}_{\mathrm{<mtext>cat</mtext>}}\text{K}{}_{\mathrm{m}}$ profile, which appears to be shifted from a higher value, pK_E2 of 9.0, for the native enzyme. The single (acid-limb) pK which characterizes the k_cat profile of the native enzyme is also found to be perturbed to a lesser extent by nitration. A kinetically competent reverse protonation mechanism, based on chemical modification and crystallographic evidence for the enzyme, is described.     

Characterization of a novel β-glucosidase from a Stachybotrys strain

An improved mutant was isolated from the cellulolytic fungus Stachybotrys sp. after nitrous acid mutagenesis. It was fed-batch cultivated on cellulose and its extracellular cellulases (mainly the endoglucanases and β-glucosidases) were analyzed. One β-glucosidase was purified to homogeneity after two steps, MonoQ and gel filtration and shown to be a dimeric protein. The molecular weight of each monomer is 85 kDa. Besides its aryl β-glucosidase activity towards salicin, methyl-umbellypheryl-β-d-glucoside (MUG) and p-nitrophenyl-β-d-glucoside (pNPG), it showed a true β-glucosidase activity since it splits cellobiose into two glucose monomers. The V_max and the K_m kinetics parameters with pNPG as substrate were 78 U/mg and 0.27 mM, respectively. The enzyme shows more affinity to pNPG than cellobiose and salicin whose apparent values of K_m were, respectively, 2.22 and 37.14 mM. This enzyme exhibits its optimal activity at pH 5 and at 50 °C. Interestingly, this activity is not affected by denaturing gel conditions (SDS and β-mercaptoethanol) as long as it is not pre-heated. The N-terminal sequence of the purified enzyme showed a significant homology with the family 1 β-glucosidases of Trichoderma reesei and Humicola isolens even though these two enzymes are much smaller in size.     

Purification and characterization of x-prolyl-dipeptidyl aminopeptidase from Lactococcus lactis subsp. cremoris NRRL 634

Summary An X-prolyl-dipeptidylaminopep tidase (Pep-XP) was purified from the crude intracellular extract of Lactococcus lactis subsp. cremoris NRRL 634 by ion exchange and gel filtration chromatographies. The enzyme was purified 80-fold with a recovery of 6%, and appeared as a single band with a molecular weight of about 80 kDa on polyacrylamide gel electrophoresis with sodium dodecyl sulphate (SDS-PAGE). The peptidase showed its maximal activity on arginyl-proline-p-nitroanilide at pH 7.0 and at a temperature of 45 °C, although there was a good activity of Pep-XP in the pH range of 5.5–7.0 and temperatures between 40 and 50 °C. The Michaelis constant (K _m) and the maximum reaction velocity (V _max) values were 0.92 mM and 7.9 U/mg protein min, respectively. The activity of Pep-XP was completely inhibited by phenylmethanesulphonyl fluoride, an inhibitor of serine peptidases, and metal chelators had little effect on enzyme activity. The purified enzyme hydrolyzed synthetic substrates whose structure is X-Pro-Y like Lys-Pro-pNA, but did not hydrolyse Pro-pNA or azocasein, showing that the enzyme did not have aminopeptidase or endopeptidase activities.     

Purification and kinetic studies on a porphobilinogen deaminase from the unicellular green alga Scenedesmus obliquus

Porphobilinogen deaminase, the enzyme condensing four molecules of porphobilinogen, was isolated and purified from light grown Scenedesmus obliquus (wild type). The purification procedure included heat treatment, ammonium sulphate fractionation, gel filtration, high-resolution anion-exchange chromatography and hydrophobic interaction chromatography. The enzyme was purified 1368-fold, compared to the initial crude extract. Its final specific activity was 6812 units · (mg · protein)^?1 at pH 7.4 with a recovery of 44%. The relative molecular mass was 33000, as determined by Sephadex G-100 gel filtration, and 35900 by lithium dodecyl sulfate-polyacrylamide-gel electrophoresis, indicating that the enzyme is a monomer. Studies of initial reaction velocities showed a linear progress curve for hydroxymethylbilane formation and a hyperbolic dependence of the initial reaction rate on substrate concentration, consistent with a sequential displacement mechanism. Apparent kinetic constants (K _m and V _max) for the conversion of porphobilinogen to hydroxymethylbilane at 37 ° C, pH 7.4, were 79 μM and 176 pmol · min^?1, respectively. Variation of both V _max and K _max with pH indicated the presence of ionizable groups in the enzyme-substrate complex(es), showing a single ionization (pK 7.15) in V _max/K _m plots. A sharp pH-profile for V _max was interpreted as a positive cooperative proton dissociation. In spite of the two pathways existing for 5-aminolevulinate biosynthesis in Scenedesmus, currently there is no indication of the existence of two porphobilinogen deaminases or even of isoenzymes.     

Terminal deoxynucleotidyl transferase: characterization of extraction and assay conditions from human and calf tissue.

Methods of extraction and assay of terminal deoxynucleotidyl transferase (TdT) from human lymphoblasts and calf thymus were compared. A high salt concentration was mandatory for complete enzyme extraction, while dialysis of the crude extract resulted in a major loss of enzyme activity. In addition, TdT was partially purified from lymphoblasts of patients with acute lymphoblastic leukemia. The K_m for the monomer, deoxy-guanosine 5′-triphosphate (dGTP), is high (~0.1 mm) in the presence of either Mg²⁺ or Mn²⁺, whereas the K_m for the initiator, poly(deoxyadenylic acid $\text{[poly(d(pA)}\text{50}\text{)]}$, with an average chain length of 50 residues, is 2.5 μm in the presence of Mg²⁺ and 0.3 μm in the presence of Mn²⁺. The maximum velocity is higher for the calf thymus TdT in the presence of Mg²⁺ than in Mn²⁺. Human TdT catalyzes the polymerization of dGTP at a higher rate in the presence of Mn²⁺ than with Mg²⁺. These data illustrate that partially purified human TdT differs in catalytic properties from the purified calf thymus enzyme. Therefore, optimal conditions for assay of TdT in extracts from calf and human tissues differ.