The proteoliposomal steady state. Effect of size, capacitance and membrane permeability on cytochrome-oxidase-induced ion gradients.

1. The flux pathways for H+ and K+ movements into and out of proteoliposomes incorporating cytochrome c oxidase have been investigated as a function of the electrical and geometrical properties of the vesicles. 2. The respiration-induced pH gradient (delta pH) and membrane potential (delta psi) are mutually dependent and individually sensitive to the permeability properties of the membrane. A lowering or abolition of delta psi by the addition of valinomycin increased the steady-state level of delta pH. Conversely, removal of delta pH by the addition of nigericin resulted in a higher steady-state delta psi. 3. Vesicles prepared by sonication followed by centrifugation maintained similar pH gradients at steady state to those in vesicles prepared by dialysis, although the time taken to reach steady state was longer. Higher pH gradients can be induced in non-centrifuged sonicated preparations. 4. No significant differences were found in H+ and K+ permeability between proteoliposomes prepared by dialysis or by sonication. The permeability coefficient of the vesicle bilayers for H+ was 6.1 x 10(-4) cm.s-1 and that for K+ was 7.5 x 10(-10) cm.s-1. An initial fast change in internal pH was seen on the addition of external acid or alkali, followed by a slower, ionophore-sensitive, change. The initial fast phase can be increased by the lipid-soluble base dibucaine and the weak acid oleate. In the absence of ionophores, increasing concentrations of oleate increased the rate of H+ translocation to a level similar to that seen in the presence of nigericin. Internal alkalinization could also be induced by oleate upon the addition of potassium sulphate. 5. The initial, pre-steady-state and steady-state delta pH and delta psi changes can be simulated using a model in which the enzyme responds to both delta pH and delta psi components of the protonmotive force. At steady state, the electrogenic entry of K+ is countered by electroneutral exit via a K+/H+ exchange. 6. The permeability coefficient, PH, calculated from H+ flux under steady-state turnover conditions, was approx. 100 times higher than the corresponding 'passive' measurements of PH. Under conditions of oxidase turnover, the vesicles appear to be intrinsically more permeable to protons.     

Reconstitution of lactic dehydrogenase. Noncovalent aggregation vs. reactivation. 2. Reactivation of irreversibly denatured aggregates

Noncovalent aggregation is a side reaction in the process of reconstitution of oligomeric enzymes (e.g., lactic dehydrogenase) after preceding dissociation, denaturation, and deactivation. The aggregation product is of high molecular weight and composed of monomers which are trapped in a minium of conformational energy different from the one characterizing the native enzyme. This energy minimum is protected by a high activation energy of dissociation such that the aggregates are perfectly stable under nondenaturing conditions, and their degradation is provided only by applying strong denaturants, e.g., 6 M guanidine hydrochloride at neutral or acidic pH. The product of the slow redissolution process is the monomeric enzyme in its random configuration, which may be reactivated by diluting the denaturant under optimum conditions of reconstitution. The yield and the kinetics of reactivation of lactic dehydrogenase from pig skeletal muscle are not affected by the preceding aggregation-degradation cycle and are independent of different modes of aggregate formation (e.g., by renaturation at high enzyme concentration or heat aggregation). The kinetics of reactivation may be described by one single rate-determining bimolecular step with k2 = 3.9 x 10(4) M-1 s-1 at zero guanidine concentration. The reactivated enzyme consists of the native tetramer, characterized by enzymatic and physical properties identical with those observed for the enzyme in its initial native state.     

The relationship between thermal stability and pH optimum studied with wild-type and mutant Trichoderma reesei cellobiohydrolase Cel7A.

The major cellulase secreted by the filamentous fungus Trichoderma reesei is cellobiohydrolase Cel7A. Its three-dimensional structure has been solved and various mutant enzymes produced. In order to study the potential use of T. reesei Cel7A in the alkaline pH range, the thermal stability of Cel7A was studied as a function of pH with the wild-type and two mutant enzymes using different spectroscopic methods. Tryptophan fluorescence and CD measurements of the wild-type enzyme show an optimal thermostability between pH 3.5-5.6 (Tm, 62 +/- 2 degrees C), at which the highest enzymatic activity is also observed, and a gradual decrease in the stability at more alkaline pH values. A soluble substrate, cellotetraose, was shown to stabilize the protein fold both at optimal and alkaline pH. In addition, unfolding of the Cel7A enzyme and the release of the substrate seem to coincide at both acidic and alkaline pH, demonstrated by a change in the fluorescence emission maximum. CD measurements were used to show that the five point mutations (E223S/A224H/L225V/T226A/D262G) that together result in a more alkaline pH optimum [Becker, D., Braet, C., Brumer, H., III, Claeyssens, M., Divne, C., Fagerstr?m, R.B., Harris, M., Jones, T.A., Kleywegt, G.J., Koivula, A., et al. (2001) Biochem. J.356, 19-30], destabilize the protein fold both at acidic and alkaline pH when compared with the wild-type enzyme. In addition, an interesting time-dependent fluorescence change, which was not observed by CD, was detected for the pH mutant. Our data show that in order to engineer more alkaline pH cellulases, a combination of mutations should be found, which both shift the pH optimum and at the same time improve the thermal stability at alkaline pH range.     

Alkaline Phosphatase and Other Hydrolyases Produced by Cenococcum graniforme, an Ectomycorrhizal Fungus

Cell extracts of Cenococcum graniforme have been found to contain the following hydrolytic enzymes: protease, esterase, alpha-d-galactopyranosidase, beta-d-galactopyranosidase, alpha-d-mannopyranosidase, beta-d-xylopyranosidase, alpha-d-glucopyranosidase, beta-d-glucopyranosidase, and alkaline phosphatase. Sulfatase, inorganic pyrophosphatase, and beta-d-mannopyranosidase were not detected in the extracts. beta-d-Xylopyranosidase and alpha-d-mannopyranosidase were most active in the neutral pH range, protease and phosphatase were most active in the alkaline pH range, and other enzymes were most active in the acidic pH range. These enzymes showed a high association with cell wall material, and the release of enzymes from the cells into the culture fluid appeared to occur only when the cells were undergoing autolysis. Alkaline phosphatase in C. graniforme is a constitutive enzyme, and examination of the alkaline phosphatase following a purification of 265-fold produced the following characteristics: pH optimum of 9.5, M(r) of 60,000, K(m) of 2.1 x 10 M for p-nitrophenylphosphate, and activation energy for hydrolysis of the substrate at 9.9 kcal (1 cal = 4.184 J)/mol.     

A pyrimidine-guanine sequence-specific ribonuclease from Rana catesbeiana (bullfrog) oocytes.

A pyrimidine-guanine sequence-specific ribonuclease (RC-RNase) was purified from Rana catesbeiana (bullfrog) oocytes by sequential phosphocellulose, Sephadex G75, heparin Sepharose CL 6B and CM-Sepharose CL 6B column chromatography. The purified enzyme with molecular weight of 13,000 daltons gave a single band on SDS-polyacrylamide gel. One CNBr-cleaved fragment has a sequence of NVLSTTRFQLNT/TRTSITPR, which is identical to residues 59-79 of a sialic acid binding lectin from R. catesbeiana eggs, and is 71% homologous to residues 60-80 of an RNase from R. catesbeaina liver. The RC-RNase preferentially cleaved RNA at pyrimidine residues with a 3' flanking guanine under various conditions. The sequence specificity of RC-RNase was further confirmed with dinucleotide as substrates, which were analyzed by thin layer chromatography after enzyme digestion. The values of kcat/km for pCpG, pUpG and pUpU were 2.66 x 10(7) M-1s-1, 2.50 x 10(7) M-1s-1 and 2.44 x 10(6) M-1s-1 respectively, however, those for other phosphorylated dinucleotides were less than 2% of pCpG and pUpG. As compared to single strand RNA, double strand RNA was relatively resistant to RC-RNase. Besides poly (A) and poly (G), most of synthetic homo- and heteropolynucleotides were also susceptible to RC-RNase. The RC-RNase was stable in the acidic (pH 2) and alkaline (pH 12) condition, but could be inactivated by heating to 80 degrees C for 15 min. No divalent cation was required for its activity. Furthermore, the enzyme activity could be enhanced by 2 M urea, and inhibited to 50% by 0.12 M NaCl or 0.02% SDS.     

Kinetic study of soybean beta-amylase. The effect of pH.

1) Beta-Amylase [EC 3.2.1.2] was prepared from defatted hawk eye soybean flour. The enzyme concentration dependence of the initial velocity for the hydrolytic reaction was investigated at pH 5.4 in the range of the enzyme concentration from 6.6 x 10(-10) M to 5.0 x 10(-6) M. It was found that the initial velocity was proportional to the enzyme concentration in this range. 2) The hydrolyses of maltodextrin (DPn = 74.4) and soluble starch catalyzed by soybean beta-amylase were investigated in the pH range from 3.0 to 9.1 at 25 degrees C, and the Michaelis constant, Km, and the maximum velocity, V, for each substrate were determined at each pH. The pH-rate profile showed a bell-shaped curve, and the pH "optimum" was at 5.85. From Dixon plots of V and V/Km, the pK values were found to be 3.5 and 8.2 for the free enzyme, and 3.5 and 8.5 for the enzyme-substrate complex. The pH-rate profile in the presence of 25% methanol (v/v) was also obtained at alkaline pH. The pKe values were the same as those in the absence of methanol. Based on these results, it was estimated that the ionizable acidic group was an amino group and the basic group was a carboxyl one.     

Kinetics studies in a continuous steady state hollow fiber membrane enzyme reactor

Porous hollow cellulose fibers have been used to separate a nonflowing enzyme solution of alkaline phosphatase from a continuous flow of substrate. The porosity of the hollow fiber membrane allows the substrate and product to diffuse freely through the membrane while restricting the permeation of the enzyme. The resulting “immobilized” enzyme system has been shown to behave as a continuous reactor—converting p-nitrophenylphosphate to p-nitrophenol. By varying the concentrations, flow rate, etc., either diffusion or enzyme kinetics can be studied. The continual influx of product and removal of substrate at steady state allows the study of kinetics of relatively short half-life enzymes and unstable systems.     

硅藻土固定中性脂肪酶的制备及其性质

以硅藻土为载体,采用吸附法,对脂肪酶进行固定化,研究了固定化条件对固定化脂肪酶的催化活性的影响,得到最佳的固定化条件：给酶量为33374U/g,固定化温度为35℃,pH值为7.5,时间为4h,此时固定化酶的活力约为5833U/g载体。固定化酶的热稳定性较游离酶有了很大的提高,其在80℃以下能保持80%以上的酶活,而游离酶60℃残余酶活仅为5%。最适反应温度和最适pH值也分别由游离酶的40℃上升至50℃和由7上升到7.5。对固定化中的中性脂肪酶在生物柴油合成中的应用也进行了初步研究。     

Immobilization of Exo-maltotetraohydrolase and Pullulanase

A dual enzyme system of exo-maltotetraohydrolase [EC 3.2.1.60] and pullulanase [EC 3.2.1.41] was studied for the continuous production of maltotetraose. Porous chitosan beads were selected from among many carriers as the best carrier to immobilize both enzymes.

The properties of the immobilized enzymes were examined and compared with those of the native enzymes. For exo-maltotetraohydrolase, the optimum pH of the immobilized enzyme shifted slightly to the acidic side and the pH stability was improved on the alkaline side. The optimum temperature of the immobilized enzyme increased by about 15°C and thermostability was improved by about 10°C. As for pullulanase, very little difference in thermostability was observed.

The effects of operating conditions on the continuous production of maltotetraose using exo- maltotetraohydrolase immobilized on the porous chitosan beads were examined. Porous chitosan beads were recognized to be superior to Diaion HP-50.

The continuous production of maltotetraose was accomplished using the dual immobilized enzyme system. The dual enzyme system proved to be effective to increase the maltotetraose content in the product. A stable operation was successfully continued for more than 60 days.     

Bovine kidney alkaline phosphatase. Catalytic properties, subunit interactions in the catalytic process, and mechanism of Mg2+ stimulation.

Kidney alkaline phosphatase is an enzyme which requires two types of metals for maximal activity: zinc, which is essential, and magnesium, which is stimulatory. The main features of the Mg2+ stimulation have been analyzed. The stimulation is pH-dependent and is observed mainly between pH 7.5 and 10.5. Mg2+ binding to native alkaline phosphatase is characterized by a dissociation constant of 50 muM at pH 8.5,25 degrees. Binding of Zn2+ is an athermic process. Both the rate constants of association, ka, and of dissociation, kd, have low values. Typical values are 7 M(-1) at pH 8.0, 25 degrees, for ka and 4.10(-4) S(-1) at pH 8.0, 25 degrees, for kd. The on and off processes have high activation energies of 29 kcal mol (-1). Mg2+ can be replaced at its specific site by Mn2+, Co2+, Ni2+, and Zn2+. Zinc binding to the Mg2+ site inhibits the native alkaline phosphatase. Mn2+, Co2+, and Ni2+ also bind to the Mg2+ site with a stimulatory effect which is nearly identic-al with that of Mg2+, Mn2+ is the stimulatory cation which binds most tightly to the Mg2+ site; the dissociation constant of the Mn2+ kidney phosphatase complex is 2 muM at pH 8.5. The stoichiometry of Mn2+ binding has been found to be 1 eq of Mn2+ per mol of dimeric kidney phosphatase. The native enzyme displays absolute half-site reactivity for Mn2+ binding. Mg2+ binding site and the substrate binding sites are distinct sites. The Mg2+ stimulation corresponds to an allosteric effect. Mg2+ binding to its specific sites does not affect substrate recognition, it selectively affects Vmax values. Quenching of the phosphoenzyme formed under steady state conditions with [32P]AMP as a substrate as well as stopped flow analysis of the catalyzed hydrolysis of 2,4-dinitrophenyl phosphate or p-nitrophenyl phosphate have shown that the two active sites of the native and of the Mg2+-stimulated enzyme are not equivalent. Stopped flow analysis indicated that one of the two active sites was phosphorylated very rapidly whereas the other one was phosphorylated much more slowly at pH 4.2. Half of the sites were shown to be reactive at pH 8.0. Quenching experiments have shown that only one of the two sites is phosphorylated at any instant; this result was confirmed by the stopped flow observation of a burst of only 1 mol of nitrophenol per mol of dimeric phosphatase in the pre-steady state hydrolysis of p-nitrophenyl phosphate. The half-of-the-sites reactivity observed for the native and for the Mg2+-stimulated enzyme indicates that the same type of complex, the monophosphorylated complex, accumulates under steady state conditions with both types of enzymes. Mg2+ binding to the native enzyme at pH 8.0 increases considerably the dephosphorylation rate of this monophosphorylated intermediate. A possible mechanism of Mg2+ stimulation is discussed.     

Mutationally altered rate constants in the mechanism of alkaline phosphatase

The hydrolysis of phosphate esters by a mutationally altered alkaline phosphatase from Escherichia coli was studied by both steady-state and transient-kinetic methods. The difference between the catalytic-centre activities of the mutationally altered and the wild-type alkaline phosphatases was found to vary with pH and at optimal pH values the modified enzyme had the higher activity. Stopped-flow experiments at acidic pH values showed that transient product formation by the mutationally altered enzyme was faster than that with the wild-type enzyme whereas the rate of the steady state was slower. In the alkaline pH region, the transient was observed in the reaction of only the modified enzyme and not the wild type. These observations permit a fuller characterization of the individual steps in the catalytic mechanism of alkaline phosphatase than is possible by study of only the wild-type enzyme.     

Control of electron transfer by the electrochemical potential gradient in cytochrome-c oxidase reconstituted into phospholipid vesicles

The kinetics of electron transfer between cytochrome-c oxidase and ruthenium hexamine has been characterized using the native enzyme or its cyanide complex either solubilized by detergent (soluble cytochrome oxidase) or reconstituted into artificial phospholipid vesicles (cytochrome oxidase-containing vesicles). Ru(NH3)2+6 (Ru(II] reduces oxidized cytochrome a, following (by-and-large) bimolecular kinetics; the second order rate constant using the cyanide complex of the enzyme is 1.5 x 10(6) M-1 s-1, for the enzyme in detergent, and slightly higher for COV. In the case of COV the kinetics are not affected by the addition of ionophores. Upon mixing fully reduced cytochrome oxidase with oxygen (in the presence of excess reductants), the oxidation leading to the pulsed enzyme is followed by a steady state phase and (eventually) by complete re-reduction. When the concentrations of dioxygen and oxidase are sufficiently low (micromolar range), the time course of oxidation can be resolved by stopped flow at room temperature, yielding an apparent bimolecular rate constant of 5 x 10(7) M-1 s-1. After exhaustion of oxygen and end of steady state, re-reduction of the pulsed enzyme by the excess Ru(II) is observed; the concentration dependence shows that the rate of re-reduction is limited at 3 s-1 in detergent; this limiting value is assigned to the intramolecular electron transfer process from cytochrome a-Cua to the binuclear center. Using the reconstituted enzyme, the internal electron transfer step is sensitive to ionophores, increasing from 2-3 to 7-8 s-1 upon addition of valinomycin and carbonyl cyanide m-chlorophenylhydrazone. This finding indicates for the first time an effect of the electrochemical potential across the membrane on the internal electron transfer rate; the results are compared with expectations based on the hypothesis formulated by Brunori et al. (Brunori, M., Sarti, P., Colosimo, A., Antonini, G., Malatesta, F., Jones, M.G., and Wilson, M.T. (1985) EMBO J. 4, 2365-2368), and their bioenergetic relevance is discussed with reference to the proton pumping activity of the enzyme.     

A novel intermediate in the interaction of thiosemicarbazide with sheep liver serine hydroxymethyltransferase.

An unusual intermediate bound to the enzyme was detected in the interaction of thiosemicarbazide with sheep liver serine hydroxymethyltransferase. This intermediate had absorbance maxima at 464 and 440 nm. Such spectra are characteristic of resonance stabilized intermediates detected in the interaction of substrates and quasi-substrates with pyridoxal phosphate enzymes. An intermediate of this kind has not been detected in the interaction of thiosemicarbazide with other pyridoxal phosphate enzymes. This intermediate was generated slowly (t 1/2 = 4 min) following the addition of thiosemicarbazide (200 microM) to sheep liver serine hydroxymethyltransferase (5 microM). It was bound to the enzyme as evidenced by circular dichroic bands at 464 and 440 nm and the inability to be removed upon Centricon filtration. The kinetics of interaction revealed that thiosemicarbazide was a slow binding reversible inhibitor in this phase with a k(on) of 11 M-1 s-1 and a k(off) of 5 x 10(-4) s-1. The intermediate was converted very slowly (k = 4 x 10(-5) s-1) to the final products, namely the apoenzyme and the thiosemicarbazone of pyridoxal phosphate. A minimal kinetic mechanism involving the initial conversion to the intermediate absorbing at longer wavelengths and the conversion of this intermediate to the final product, as well as, the formation of pyridoxal phosphate-thiosemicarbazone directly by an alternate pathway is proposed.     

Enhanced stabilization of mungbean thiol protease immobilized on glutaraldehyde-activated chitosan beads

A thiol protease purified from mungbean seedlings was immobilized on chitosan beads cross-linked with glutaraldehyde. The yield of the immobilized enzyme was maximum (～99%) at 1% concentration each of chitosan and glutaraldehyde. The immobilized enzyme showed reusability for 15 batch reactions. Immobilization shifted the optimum pH of the enzyme to a more acidic range and enhanced its stability both at acidic as well as alkaline pH values compared to the free enzyme. The stability of the enzyme to temperature and in aqueous non-conventional medium (ethanol and DMSO) was significantly improved by the immobilization process. The immobilized enzyme exhibited mass transfer limitation reflected by a higher apparent K_m value. This study produced an immobilized biocatalyst having improved characteristics and better operational stability than the soluble enzyme. The increase in stability in the presence of high concentrations of ethanol and DMSO may make it useful for catalyzing organic reactions such as trans-esterification and trans-amidation similar to other cysteine proteinases.     

Immobilization and stabilization of α-galactosidase on Sepabeads EC-EA and EC-HA

α-Galactosidase from tomato has been immobilized on Sepabead EC-EA and Sepabead EC-HA, which were activated with ethylendiamino and hexamethylenediamino groups, respectively. Two strategy was used for the covalent immobilization of α-galactosidase on the aminated Sepabeads: covalent immobilization of enzyme on glutaraldehyde activated support and cross-linking of the adsorbed enzymes on to the support with glutaraldehyde. By using these two methods, all the immobilized enzymes retained very high activity and the stability of the enzyme was also improved. The obtained results showed that, the most stable immobilized α-galactosidase was obtained with the second strategy. The immobilized enzymes were characterized with respect to free counterpart. Some parameters effecting to the enzyme activity and stability were also analyzed. The optimum temperature and pH were found as 60 °C and pH 5.5 for all immobilized enzymes, respectively. All the immobilized α-galactosidases were more thermostable than the free enzyme at 50 °C. The stabilities of the Sepabead EC-EA and EC-HA adsorbed enzymes treated with glutaraldehyde compared to the stability of the free enzyme were a factor of 6 for Sepabead EC-EA and 5.3 for Sepabead EC-HA. Both the free and immobilized enzymes were very stable between pH 3.0 and 6.0 and more than 85% of the initial activities were recovered. Under the identical storage conditions the free enzyme lost its initial activity more quickly than the immobilized enzymes at the same period of time. The immobilized α-galactosidase seems to fulfill the requirements for different industrial applications.     

Expression of spinach glycolate oxidase in Saccharomyces cerevisiae: purification and characterization.

Glycolate oxidase from spinach has been expressed in Saccharomyces cerevisiae. The active enzyme was purified to near-homogeneity (purification factor approximately 1400-fold) by means of hydroxyapatite and anion-exchange chromatography. The purified glycolate oxidase is nonfluorescent and has absorbance peaks at 448 (epsilon = 9200 M-1 cm-1) and 346 nm in 0.1 M phosphate buffer, pH 8.3. The large bathochromic shift of the near-UV band indicates that the N(3) position is deprotonated at pH 8.3. A pH titration revealed that the pK of the N(3) is shifted from 10.3 in free flavin to 6.4 in glycolate oxidase. Glycolate oxidase is competitively inhibited by oxalate with a Kd of 0.24 mM at 4 degrees C in 0.1 M phosphate buffer, pH 8.3. Three pieces of evidence demonstrate that glycolate oxidase stabilizes a negative charge at the N(1)-C(2 = O) locus: the enzyme forms a tight sulfite complex with a Kd of 2.7 x 10(-7) M and stabilizes the anionic flavosemiquinone and the benzoquinoid form of 8-mercapto-FMN. Steady-state analysis at pH 8.3, 4 degrees C, yielded a Km = 1 x 10(-3) M for glycolate and Km = 2.1 x 10(-4) M for oxygen. The turnover number has been determined to be 20 s-1. Stopped-flow studies of the reductive (k = 25 s-1) and oxidative (k = 8.5 x 10(4) M-1 s-1) half-reactions have identified the reduction of glycolate oxidase to be the rate-limiting step.     

Acid inactivation of short-lived rat liver enzymes.

The stabilities of nine rat liver cytosol enzymes were compared at a variety of pH values. The cytosol enzymes studied were (a) those with half-lives in vivo of 3 days or longer: lactate dehydrogenase, arginase, glyceraldehyde phosphate dehydrogenase and alanine aminotransferase, (b) those with half-lives in vivo shorter than 2 days; glucokinase, dihydroorotase, serine dehydratase and tyrosine aminotransferase and (c) catalase, which has an intermediate half-life of 2.5 days for the protein protion. All the enzymes were stable in vitro at neurtal and alkaline pH values. However, at acidic pH values (pH 4): the long-lived enzymes (a) were stable; the short-lived enzymes (b) were completely inactivated with one exception; and catalase was partially inactivated. Tyrosine aminotransferase was the exception in that it is a short-lived enzyme in vivo but stable under all conditions tested in vitro. The finding that long-lived enzymes are stable in an acid milieu and short-lived enzymes are generally unstable was only observed if certain ligands (NAD+, pyridoxal 5'-phosphate, Mn2+, amino acids) were added to the invitro system. Lysosomal extracts did not accelerate the rate of inactivation of any cytosol enzyme in acidic solutions. These results indicate that if degradation of intracellular enzymes occurs in lysosomes, acid inactivation and denaturation of enzymes may be the initial event in determining the functional half-lives of the enzymes in vivo.     

Kinetic mechanism of cytosine DNA methyltransferase MspI.

A kinetic analysis of MspI DNA methyltransferase (M.MspI) is presented. The enzyme catalyzes methylation of lambda-DNA, a 50-kilobase pair linear molecule with multiple M.MspI-specific sites, with a specificity constant (kcat/KM) of 0.9 x 10(8) M-1 s-1. But the values of the specificity constants for the smaller DNA substrates (121 and 1459 base pairs (bp)) with single methylation target or with multiple targets (sonicated lambda-DNA) were less by an order of magnitude. Product inhibition of the M.MspI-catalyzed methylation reaction by methylated DNA is competitive with respect to DNA and noncompetitive with respect to S-adenosylmethionine (AdoMet). The S-adenosylhomocysteine inhibition of the methylation reaction is competitive with respect to AdoMet and uncompetitive with respect to DNA. The presteady state kinetic analysis showed a burst of product formation when AdoMet was added to the enzyme preincubated with the substrate DNA. The burst is followed by a constant rate of product formation (0.06 mol per mol of enzyme s-1) which is similar to catalytic constants (kcat = approximately 0.056 s-1) measured under steady state conditions. The isotope exchange in chasing the labeled methyltransferase-DNA complex with unlabeled DNA and AdoMet leads to a reduced burst as compared with the one involving chase with labeled DNA and AdoMet. The enzyme is capable of exchanging tritium at C-5 of target cytosine in the substrate DNA in the absence of cofactor AdoMet. The kinetic data are consistent with an ordered Bi Bi mechanism for the M.MspI-catalyzed DNA methylation where DNA binds first.     

Preparation and properties of proteases immobilized on anion exchange resin with glutaraldehyde

High activity alkaline protease was obtained when the enzyme was immobilized on Dowex MWA-1 (mesh 20–50) with 10% glutaraldehyde in chilled phosphate buffer (M/15, pH 6.5). Activity yields of the protease and rennet were 27 and 29, respectively. The highest activities appeared at 60°C, pH 10 for alkaline protease and 50°C, pH 4.0 for rennet. The properties of both proteases were not essentially changed by the immobilization except that the K_m values of both enzymes were increased about tenfold as a result of immobilization. Both proteases in the immobilized state were more stable than those in the free state at 60°C. Other peptide hydrolases, β-galactosidase, invertase, and glucoamylase, were successfully immobilized with high activities, but lipase, hexokinase, glucose-6-phosphate dehydrogenase, and xanthine oxidase became inactive.     

Chemical and transient state kinetic studies on the formation and decomposition of horseradish peroxidase compounds XI and XII

Previous studies on the chlorination reaction catalyzed by horseradish peroxidase using chlorite as the source of chlorine detected the formation of a chlorinating intermediate that was termed Compound X (Shahangian, S., and Hager, L.P. (1982) J. Biol. Chem. 257, 11529-11533). These studies indicated that at pH 10.7, the optical absorption spectrum of Compound X was similar to the spectrum of horseradish peroxidase Compound II. Compound X was shown to be quite stable at alkaline pH values. This study was undertaken to examine the relationship between the oxidation state of the iron protoporphyrin IX heme prosthetic group in Compound X and the chemistry of the halogenating intermediate. The experimental results show that the optical absorption properties and the oxidation state of the heme prosthetic group in horseradish peroxidase are not directly related to the presence of the activated chlorine atom in the intermediate. The oxyferryl porphyrin heme group in alkaline Compound X can be reduced to a ferric heme species that still retains the activated chlorine atom. Furthermore, the reaction of chlorite with horseradish peroxidase at acidic pH leads to the secondary formation of a green intermediate that has the spectral properties of horseradish peroxidase Compound I (Theorell, H. (1941) Enzymologia 10, 250-252). The green intermediate also retains the activated chlorine atom. By analogy to peroxidase Compound I chemistry, the heme prosthetic group in the green chlorinating intermediate must be an oxyferryl porphyrin pi-cation radical species (Roberts, J. E., Hoffman, B. M., Rutter, R. J., and Hager, L. P. (1981) J. Am. Chem. Soc. 103, 7654-7656). To be consistent with traditional peroxidase nomenclature, the red alkaline form of Compound X has been renamed Compound XII, and the green acidic form has been named Compound XI. The transfer of chlorine from the chlorinating intermediate to an acceptor molecule follows an electrophilic (rather than a free radical) path. A mechanism for the reaction is proposed in which the activated chlorine atom is bonded to a heteroatom on an active-site amino acid side chain. Transient state kinetic studies show that the initial intermediate, Compound XII, is formed in a very fast reaction. The second-order rate constant for the formation of Compound XII is approximately 1.1 x 10(7) M-1 s-1. The rate of formation of Compound XII is strongly pH-dependent. At pH 9, the second-order rate constant for the formation of Compound XII drops to 1.5 M-1 s-1. At acidic pH values, Compound XII undergoes a spontaneous first-order decay to yield Compound XI.(ABSTRACT TRUNCATED AT 400 WORDS)