Single amino acid mutations interchange the reaction specificities of cyclodextrin glycosyltransferase and the acarbose-modifying enzyme acarviosyl transferase

Acarviosyl transferase (ATase) from Actinoplanes sp. SE50/110 is a bacterial enzyme that transfers the acarviosyl moiety of the diabetic drug acarbose to sugar acceptors. The enzyme exhibits 42% sequence identity with cyclodextrin glycosyltransferases (CGTase), and both enzymes are members of the alpha-amylase family, a large clan of enzymes acting on starch and related compounds. ATase is virtually inactive on starch, however. In contrast, ATase is the only known enzyme to efficiently use acarbose as substrate (2 micromol min(-1) mg(-1)); acarbose is a strong inhibitor of CGTase and of most other alpha-amylase family enzymes. This distinct reaction specificity makes ATase an interesting enzyme to investigate the variation in reaction specificity of alpha-amylase family enzymes. Here we show that a G140H mutation in ATase, introducing the typical His of the conserved sequence region I of the alpha-amylase family, changed ATase into an enzyme with 4-alpha-glucanotransferase activity (3.4 micromol min(-1) mg(-1)). Moreover, this mutation introduced cyclodextrin-forming activity into ATase, converting 2% of starch into cyclodextrins. The opposite experiment, removing this typical His side chain in CGTase (H140A), introduced acarviosyl transferase activity in CGTase (0.25 micromol min(-1) mg(-1)).     

Reactions of the class II peroxidases, lignin peroxidase and Arthromyces ramosus peroxidase, with hydrogen peroxide. Catalase-like activity, compound III formation, and enzyme inactivation

The reactions of the fungal enzymes Arthromyces ramosus peroxidase (ARP) and Phanerochaete chrysosporium lignin peroxidase (LiP) with hydrogen peroxide (H(2)O(2)) have been studied. Both enzymes exhibited catalase activity with hyperbolic H(2)O(2) concentration dependence (K(m) approximately 8-10 mm, k(cat) approximately 1-3 s(-1)). The catalase and peroxidase activities of LiP were inhibited within 10 min and those of ARP in 1 h. The inactivation constants were calculated using two independent methods; LiP, k(i) approximately 19 x 10(-3) s(-1); ARP, k(i) approximately 1.6 x 10(-3) s(-1). Compound III (oxyperoxidase) was detected as the majority species after the addition of H(2)O(2) to LiP or ARP, and its formation was accompanied by loss of enzyme activity. A reaction scheme is presented which rationalizes the turnover and inactivation of LiP and ARP with H(2)O(2). A similar model is applicable to horseradish peroxidase. The scheme links catalase and compound III forming catalytic pathways and inactivation at the level of the [compound I.H(2)O(2)] complex. Inactivation does not occur from compound III. All peroxidases studied to date are sensitive to inactivation by H(2)O(2), and it is suggested that the model will be generally applicable to peroxidases of the plant, fungal, and prokaryotic superfamily.     

Allicin disrupts the cell's electrochemical potential and induces apoptosis in yeast

The volatile substance allicin gives crushed garlic (Allium sativum) its characteristic odor and is a pro-oxidant that undergoes thiol-disulfide exchange reactions with -SH groups in proteins and glutathione. The antimicrobial activity of allicin is suspected to be due to the oxidative inactivation of essential thiol-containing enzymes. We investigated the hypothesis that at threshold inhibitory levels allicin can shunt yeast cells into apoptosis by altering their overall redox status. Yeast cells were treated either with chemically synthesized, pure allicin or with allicin in garlic juice. Allicin-dependent cell oxidation was demonstrated with a redox-sensitive GFP construct and the shift in cellular electrochemical potential (E(hc)) from less than -215 to -181mV was calculated using the Nernst equation after the glutathione/glutathione disulfide couple (2GSH/GSSG) in the cell was quantified. Caspase activation occurred after allicin treatment, and yeast expressing a human antiapoptotic Bcl-XL construct was rendered more resistant to allicin. Also, a yeast apoptosis-inducing factor deletion mutant was more resistant to allicin than wild-type cells. We conclude that allicin in garlic juice can activate apoptosis in yeast cells through its oxidizing properties and that this presents an alternative cell-killing mechanism to the previously proposed specific oxidative inactivation of essential enzymes.     

A spectrophotometric assay for allicin,alliin, and alliinase (alliin lyase) with a chromogenic thiol: reaction of 4-mercaptopyridine with thiosulfinates

Allicin (diallylthiosulfinate) is the best known active compound of garlic. It is generated upon the interaction of the nonprotein amino acid alliin with the enzyme alliinase (alliin lyase, EC 4.4.1.4). Previously, we described a simple spectrophotometric assay for the determination of allicin and alliinase activity, based on the reaction between 2-nitro-5-thiobenzoate (NTB) and allicin. This reagent is not commercially available and must be synthesized. In this paper we describe the quantitative analysis of alliin and allicin, as well as of alliinase activity with 4-mercaptopyridine (4-MP), a commercially available chromogenic thiol. The assay is based on the reaction of 4-MP (lambda(max)=324nm) with the activated disulfide bond of thiosulfinates -S(O)-S-, forming the mixed disulfide, 4-allylmercaptothiopyridine, which has no absorbance at this region. The structure of 4-allylmercaptothiopyridine was confirmed by mass spectrometry. The method was used for the determination of alliin and allicin concentrations in their pure form as well as of alliin and total thiosulfinates concentrations in crude garlic preparations and garlic-derived products, at micromolar concentrations. The 4-MP assay is an easy, sensitive, fast, noncostly, and highly efficient throughput assay of allicin, alliin, and alliinase in garlic preparations.     

Stability and reactivity of acid phosphatase immobilized on composite beads of chitosan and ZrO2 powders

Equal weights of chitosan and ZrO2 powders were mixed in acetic acid solution to prepare the composite beads. They were then cross-linked with glutaraldehyde and stored with and without freeze-drying before use. The physicochemical properties of acid phosphatase immobilized on four types of the supports (wet/dried pure chitosan beads, wet/dried chitosan-ZrO2 composite beads) were compared. Various parameters including glutaraldehyde concentration, cross-linking time, enzyme concentration, temperature, and pH on enzyme activity were studied. It was shown that the activity yield of enzyme immobilized on the dried chitosan-ZrO2 beads was the highest, and the relative activity remained above 83.2% within pH 2.9-5.8. Regardless of wet or dried beads, the Michaelis constant KM and maximum rate of reaction Vmax of acid phosphatase immobilized on chitosan-ZrO2 composite beads were 1.8 times larger than those on pure chitosan beads. Of the four immobilized enzymes, the use of wet chitosan-ZrO2 bead as the support showed the lowest thermal deactivation energy (78 kJ mol(-1)).     

大蒜素激活SIRT1通路抑制过氧化氢诱导人脐静脉内皮细胞的衰老

Sirtuin1(SIRT1)活性的异常与血管内皮细胞的衰老密切相关。大蒜素作为一种生物活性分子具有抗氧化、抗炎及调脂作用,然而目前尚未见关于大蒜素与SIRT1的活性调节的报道。本研究旨在阐明大蒜素对过氧化氢（H2O2）诱导人脐静脉内皮细胞（HUVECs）衰老的影响,以及Sirtuin1(SIRT1)在其中的作用。SA-β-gal染色及活性氧检测提示,与对照组相比,大蒜素明显减少H2O2诱导半乳糖苷酶阳性细胞数及活性氧的产生。用Western印迹、MTT、RT-PCR及SIRT1活化检测对SIRT1、p-SIRT1、PAI-1的蛋白质、SIRT1mRNA表达及细胞活力进行检测,结果显示,大蒜素可以逆转H2O2诱导的PAI-1表达的升高、SIRT1磷酸化及活性的降低,并且上调细胞的活力。当采用SIRT1抑制剂NAM处理后,大蒜素的这些作用均被阻断。以上结果表明,大蒜素通过激活SIRT1抑制H2O2诱导的HUVECs ROS的产生、活力的下降及细胞的衰老。     

A novel antioxidant mechanism of ebselen involving ebselen diselenide,a substrate of mammalian thioredoxin and thioredoxin reductase

The antioxidant mechanism of ebselen involves recently discovered reductions by mammalian thioredoxin reductase (TrxR) and thioredoxin (Trx) forming ebselen selenol. Here we describe a previously unknown reaction; ebselen reacts with its selenol forming an ebselen diselenide with a rate constant of 372 m(-1)s(-1). The diselenide also was a substrate of TrxR forming the selenol with K(m) of 40 microm and k(cat) of 79 min(-1) (k(cat)/K(m) of 3.3 x 10(4) m(-1)s(-1)). Trx increased the reduction because of its fast reaction with diselenide (rate constant 1.7 x 10(3) m(-1)s(-1)). Diselenide stimulated the H2O2 reductase activity of TrxR, even more efficiently with Trx present. Because the mechanism of ebselen as an antioxidant has been assumed to involve glutathione peroxidase-like activity, we compared the H2O2 reductase activity of ebselen with the GSH and Trx systems. TrxR at 50 nm, far below the estimated physiological level, gave 8-fold higher activity compared with 1 mm GSH; addition of 5 microm Trx increased this difference to 13-fold. The rate constant of ebselen selenol reacting with H2O2 was estimated to be faster than 350 m(-1)s(-1). We propose novel mechanisms for ebselen antioxidant action involving ebselen selenol and diselenide formation, with the thioredoxin system rather than glutathione as the predominant effector and target.     

Formation and conversion of oxygen metabolites by Lactococcus lactis subsp. lactis ATCC 19435 under different growth conditions

A semidefined medium based on Casamino Acids allowed Lactococcus lactis ATCC 19435 to grow in the presence of oxygen at a slow rate (0.015 h(-1)). Accumulation of H(2)O(2) in the culture prevented a higher growth rate. Addition of asparagine to the medium increased the growth rate, whereby H(2)O(2) accumulated only temporarily during the lag phase. H(2)O(2) is an inhibitor for several glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase being the most sensitive. Strain ATCC 19435 contained NADH oxidase (maximum specific rate under aerobic conditions, 426 nmol of NADH min(-1) mg of protein(-1)), which reduced oxygen to water, whereby superoxide was formed as a by-product. H(2)O(2) originated from the dismutation of superoxide by superoxide dismutase. Although H(2)O(2) was rapidly destroyed under high metabolic fluxes, neither NADH peroxidase nor any other enzymatic H(2)O(2)-reducing activity was detected. However, pyruvate, the end product of glycolysis, reacted nonenzymatically and rapidly with H(2)O(2) and hence was a potential alternative for scavenging of this oxygen metabolite intracellularly. Indeed, intracellular concentrations of up to 93 mM pyruvate were detected in aerobic cultures growing at high rates. It is hypothesized that self-generated pyruvate may serve to protect L. lactis strain ATCC 19435 from H(2)O(2).     

Hemes and hemoproteins. 5: Kinetics of the peroxidatic activity of microperoxidase-8: model for the peroxidase enzymes

The peroxidatic activity of the heme octapeptide from cytochrome c, microperoxidase-8 (MP-8), was assayed at 25 degrees C under conditions where formation of Compound I is rate limiting. In the pH range 6-9, the reaction rate increased linearly with a slope close to unity. The active form of the substrate is the hydroperoxide anion, HO2-, and an extrapolated second-order rate constant was obtained for the reaction of aquoMP-8 with HO2- of 3.7 X 10(8) M-1 sec-1, which is close to the second-order rate constants reported for reaction of the peroxidase enzymes with H2O2. Comparison with published data shows that the Fe3+ ion of MP-8 reacts as expected with simple anions, electrons, and HO2-, while the analogous reactions of the enzymes all show a requirement for one H+. We conclude that the peroxidase enzymes activate H2O2 under physiological conditions through a pH-independent, H+-coupled binding of the required H2O2-. The peroxidase activity of MP-8 can be increased more than tenfold by the presence of the guanidinium ion, which is ascribed to formation of the ion-pair GuaH+HO2-; this suggests a role for the invariant distal Arg in the enzymes.     

Characterization of a beta-fructofuranosidase from Schwanniomyces occidentalis with transfructosylating activity yielding the prebiotic 6-kestose

beta-Fructofuranosidases are powerful tools in industrial biotechnology. We have characterized an extracellular beta-fructofuranosidase from the yeast Schwanniomyces occidentalis. The enzyme shows broad substrate specificity, hydrolyzing sucrose, 1-kestose, nystose and raffinose, with different catalytic efficiencies (k(cat)/K(m)). Although the main reaction catalysed by this enzyme is sucrose hydrolysis, it also produces two fructooligosaccharides (FOS) by transfructosylation. A combination of (1)H, (13)C and 2D-NMR techniques shows that the major product is the prebiotic trisaccharide 6-kestose. The 6-kestose yield obtained with this beta-fructofuranosidase is, to our concern, higher than those reported with other 6-kestose-producing enzymes, both at the kinetic maximum (76gl(-1)) and at reaction equilibrium (44gl(-1)). The total FOS production in the kinetic maximum was 101gl(-1), which corresponded to 16.4% (w/w) referred to the total carbohydrates in the reaction mixture.     

Enzymatic hydrolysis and physical characterization of commercial celluloses and cellulose-based ion-exchange powdered mixed resins

Commercial celluloses (BH20, Epicote, FC+) and their cellulose-containing powdered mixed resins (PMR) were evaluated using enzymatic and physical methods. Samples were hydrolyzed with purified Trichoderma viride cellulase extract and measured for released reducing sugar using the dinitrosalicylic acid method. Physical characterization was performed with gross specific surface areas (GSSA) and relative crystalline indices (RCI). In addition, FC+ was exposed to physical and chemical processing commonly encountered in spent PMR processing to determine potential effects on reducing sugar release in high intensity containers. Reducing sugar released from the celluloses by T. viride cellulase ranged from 135.37 to 244.48 mg day(-1); the celluloses were highly crystalline, ranging from 82.47 to 84.57%; and the GSSA medians for the celluloses ranged from 1,298.60 cm(2) g(-1) to 2,493.20 cm(2) g(-1). Most processing treatments on the FC+ reduced the amount of reducing sugar released and increased RCI. Cellulose hydrolysis rates did not show a strong correlation with the physical characterization. These results suggest that (1) celluloses and PMR can serve as abundant sources of bioavailable carbon in water treatment systems, and (2) the use of correlative physical characteristics to evaluate a cellulose-based commercial product may not accurately predict microbial activity; a complementary microbial test such as cellulose hydrolysis with cellulase may prove useful.     

3H]Allicin: preparation and applications

Allicin (diallylthiosulfinate), the active substance of garlic, has been shown to possess a variety of biological activities. Mechanistic and pharmacokinetic studies of allicin and its derivatives raise the need for a labeled compound. However, labeling of this volatile and unstable liquid requires delicate handling. Here, we describe a simple method for the preparation of (3)H-labeled allicin. This was achieved by applying synthetic [(3)H]alliin ([2,3-(3)H]allylcysteine sulfoxide) to a column containing immobilized alliinase [EC 4.1.1.4.] from garlic. Purification of [(3)H]allicin was done by differential adsorbtion of the reaction components on a neutral polystyrene resin, Porapak Q. Thiol-containing compounds are known to be the main target of allicin. In this work we demonstrated that [(3)H]allicin can be used for the synthesis of labeled [(3)H]allylmercapto derivatives of SH peptides and proteins. Thus, we prepared [(3)H]S-allylmercaptoglutathione which can be used in metabolic studies. Moreover, we showed that incubation of alliinase with [(3)H]allicin led to modification of 1.4 cysteine residues per subunit of the enzyme.     

Hydroxyl-radical production in physiological reactions. A novel function of peroxidase.

Peroxidases catalyze the dehydrogenation by hydrogen peroxide (H2O2) of various phenolic and endiolic substrates in a peroxidatic reaction cycle. In addition, these enzymes exhibit an oxidase activity mediating the reduction of O2 to superoxide (O2.-) and H2O2 by substrates such as NADH or dihydroxyfumarate. Here we show that horseradish peroxidase can also catalyze a third type of reaction that results in the production of hydroxyl radicals (.OH) from H2O2 in the presence of O2.-. We provide evidence that to mediate this reaction, the ferric form of horseradish peroxidase must be converted by O2.- into the perferryl form (Compound III), in which the haem iron can assume the ferrous state. It is concluded that the ferric/perferryl peroxidase couple constitutes an effective biochemical catalyst for the production of .OH from O2.- and H2O2 (iron-catalyzed Haber-Weiss reaction). This reaction can be measured either by the hydroxylation of benzoate or the degradation of deoxyribose. O2.- and H2O2 can be produced by the oxidase reaction of horseradish peroxidase in the presence of NADH. The .OH-producing activity of horseradish peroxidase can be inhibited by inactivators of haem iron or by various O2.- and .OH scavengers. On an equimolar Fe basis, horseradish peroxidase is 1-2 orders of magnitude more active than Fe-EDTA, an inorganic catalyst of the Haber-Weiss reaction. Particularly high .OH-producing activity was found in the alkaline horseradish peroxidase isoforms and in a ligninase-type fungal peroxidase, whereas lactoperoxidase and soybean peroxidase were less active, and myeloperoxidase was inactive. Operating in the .OH-producing mode, peroxidases may be responsible for numerous destructive and toxic effects of activated oxygen reported previously.     

Intrinsic activity of guanosine 3',5'-monophosphate-dependent protein kinase similar to adenosine 3',5'-monophosphate-dependent protein kinase. I. Phosphorylation of histone fractions.

Guanosine 3',5'-monophosphate (cyclic GMP)-dependent protein kinase partially purified from silkworm pupae reacts preferentially with H1, H2A, and H2B histones but not with H3 AND H4 histones. However, the latter can serve as substrates in the presence of a stimulatory modulator as described by Kuo and Kuo (J. Biol. Chem. 251, 4283-4286 (1976)). With H2B histone as substrate high Mg2+ concentrations (50-100 mM) are necessary for the maximum rate of reaction. Although effects of the modulator and Mg2+ vary significantly with the histone fractions employed, analysis on the phosphorylation of histone fractions provides evidence that cyclic GMP-dependent protein kinase possesses an intrinsic activity that is similar to that of adenosine 3',5'-monophosphate-dependent protein kinase.     

Antibacterial activity of snake, scorpion and bee venoms: a comparison with purified venom phospholipase A2 enzymes

AIMS: Venoms of snakes, scorpions, bees and purified venom phospholipase A(2) (PLA(2)) enzymes were examined to evaluate the antibacterial activity of purified venom enzymes as compared with that of the crude venoms. METHODS AND RESULTS: Thirty-four crude venoms, nine purified PLA(2)s and two L-amino acid oxidases (LAAO) were studied for antibacterial activity by disc-diffusion assay (100 microg ml(-1)). Several snake venoms (Daboia russelli russelli, Crotalus adamanteus, Naja sumatrana, Pseudechis guttata, Agkistrodon halys, Acanthophis praelongus and Daboia russelli siamensis) showed activity against two to four different pathogenic bacteria. Daboia russelli russelli and Pseudechis australis venoms exhibited the most potent activity against Staphylococcus aureus, while the rest showed only a moderate activity against one or more bacteria. The order of susceptibility of the bacteria against viperidae venoms was -S. aureus > Proteus mirabilis > Proteus vulgaris > Enterobacter aerogenes > Pseudomonas aeruginosa and Escherichia coli. The minimum inhibitory concentrations (MIC) against S. aureus was studied by dilution method (160-1.25 microg ml(-1)). A stronger effect was noted with the viperidae venoms (20 microg ml(-11)) as compared with elapidae venoms (40 microg ml(-1)). The MIC were comparable with those of the standard drugs (chloramphenicol, streptomycin and penicillin). CONCLUSION: The present findings indicate that viperidae (D. russelli russelli) and elapidae (P. australis) venoms have significant antibacterial effects against gram (+) and gram (-) bacteria, which may be the result of the primary antibacterial components of laao, and in particular, the PLA(2) enzymes. The results would be useful for further purification and characterization of antibacterial agents from snake venoms. SIGNIFICANCE AND IMPACT OF THE STUDY: The activity of LAAO and PLA(2) enzymes may be associated with the antibacterial activity of snake venoms.     

In situ kinetic parameters of glucose-6-phosphate dehydrogenase and phosphogluconate dehydrogenase in different areas of the rat liver acinus

The reaction velocity of glucose-6-phosphate dehydrogenase (G6PDH) and phosphogluconate dehydrogenase (PGDH) was quantified with a cytophotometer by continuous monitoring of the reaction product as it was formed in liver cryostat sections from normal, young mature female rats at 37 degrees C. Control incubations were performed in media lacking both substrate and coenzyme for G6PDH activity and lacking substrate for PGDH activity. All reaction rates were non-linear but test minus control reactions showed linearity with incubation time up to 5 min using Nitro BT as final electron acceptor. End point measurements after incubation for 5 min at 37 degrees C revealed that the highest specific activity of G6PDH was present in the intermediate area (Vmax = 7.79 +/- 1.76 mumol H2 cm-3 min-1) and of PGDH in the pericentral and intermediate areas (Vmax = 17.19 +/- 1.73 mumol H2 cm-3 min-1). In periportal and pericentral areas, Vmax values for G6PDH activity were 4.48 +/- 1.03 mumol H2 cm-3 min-1) and 3.47 +/- 0.78 mumol H2 cm-3 min-1), respectively. PGDH activity in periportal areas showed a Vmax of 10.84 +/- 0.33 mumol H2 cm3 min-1. Variation of the substrate concentration for G6PDH activity yielded similar KM values of 0.17 +/- 0.07 mM, 0.15 +/- 0.13 mM and 0.22 +/- 0.11 mM in periportal, pericentral and intermediate areas, respectively. KM values of 0.87 +/- 0.12 mM in periportal and of 1.36 +/- 0.10 mM in pericentral and intermediate areas were found for PGDH activity. The significant difference between KM values for PGDH in areas within the acinus support the hypothesis that PGDH is present in the cytoplasmic matrix and in the microsomes. A discrepancy existed between KM and Vmax values determined in cytochemical assays using cryostat sections and values calculated from biochemical assays using diluted homogenates. In cytochemical assays, the natural microenvironment for enzymes is kept for the demonstration of their activity and thus may give more accurate information on enzyme reactions as they take place in vivo.     

Thermal degradation of allicin in garlic extracts and its implication on the inhibition of the in-vitro growth of Helicobacter pylori

Allicin, the main active principle related to Allium sativum chemistry, is considered to be responsible for the bacteriostatic properties of garlic. The work described here has demonstrated the direct implication of the allicin present in solvent-free garlic extracts obtained with ethanol (ethanolic garlic extract, EGE) and acetone (acetonic garlic extract, AGE) in the inhibition of the in-vitro growth of Helicobacter pylori (Hp), the bacterium responsible for serious gastric diseases such as ulcers and even gastric cancer. The evolution of allicin concentration as a function of time and temperature has been the subject of a kinetic study. The reaction order, activation energy, and preexponential factor (in accordance with Arrhenius theory) have been determined for the decomposition process of allicin in these organic media. First-order decomposition, an activation energy of 97.4 kJ/mol, and an Arrhenius preexponential factor of 8.9 x 10(10) s(-1) have been determined for allicin in EGE. For allicin in AGE the kinetic order determined was 1.5, the activation energy 184.5 kJ/mol, and the preexponential factor 3.1 x 10(24) s(-1) (mg/L)(-0.5). The presence or absence of allicin in these garlic products was found to be crucial for the inhibition of the in-vitro growth of Hp, as demonstrated by microbiological analysis for AGE. A relationship has been identified between the effectiveness and durability of the anti-Hp properties shown by AGE and the allicin content of these products. The bacteriostatic properties were active for up to 10 months if the samples were maintained at 6 degrees C.     

Esterase catalysis of substrate vapour: enzyme activity occurs at very low hydration

It has been generally accepted that enzyme activity requires a minimal hydration of about 0.2 g H2O g(-1) protein. This fits well with evidence that hydration above this level is associated with the onset of intramolecular motions. The influence of enzyme hydration on the hydrolysis of substrate by Candida rugosa Lipase B and pig liver esterase was investigated. Each enzyme was studied as a powder at various hydration levels, using vapour phase ethyl butyrate as substrate. This procedure allows the separation of those effects that are due to hydration from those arising from diffusional constraints. We found hydrolytic activity in both enzymes at all hydration levels above zero (between 0.054-0.47 and 0.029-0.60 g H2O g(-1) protein, respectively) that were investigated. The lowest hydration level investigated, <0.03 g H2O g(-1) enzyme, corresponded to a water/enzyme mole ratio of 100 and a coverage of about 10% of the enzyme surface by water molecules. The hydrolytic activity of both enzymes was dependent on protein hydration. However, since the hydrolysis of ethyl butyrate requires water as a second substrate, the absence of activity at zero hydration does not rule out the possibility of enzyme activity in the absence of water. These results suggest that the properties conferred on proteins by water, at least above 10% surface coverage (in this case corresponding to a hydration level of 0.03 g H2O g(-1) protein), are not a requirement for enzyme catalysis.     

Kinetics of interconversion of ferrous enzymes, compound II and compound III, of wild-type synechocystis catalase-peroxidase and Y249F: proposal for the catalatic mechanism

With the exception of catalase-peroxidases, heme peroxidases show no significant ability to oxidize hydrogen peroxide and are trapped and inactivated in the compound III form by H2O2 in the absence of one-electron donors. Interestingly, some KatG variants, which lost the catalatic activity, form compound III easily. Here, we compared the kinetics of interconversion of ferrous enzymes, compound II and compound III of wild-type Synechocystis KatG, the variant Y249F, and horseradish peroxidase (HRP). It is shown that dioxygen binding to ferrous KatG and Y249F is reversible and monophasic with apparent bimolecular rate constants of (1.2 +/- 0.3) x 10(5) M(-1) s(-1) and (1.6 +/- 0.2) x 10(5) M(-1) s(-1) (pH 7, 25 degrees C), similar to HRP. The dissociation constants (KD) of the ferrous-dioxygen were calculated to be 84 microm (wild-type KatG) and 129 microm (Y249F), higher than that in HRP (1.9 microm). Ferrous Y249F and HRP can also heterolytically cleave hydrogen peroxide, forming water and an oxoferryl-type compound II at similar rates ((2.4 +/- 0.3) x 10(5) M(-1) s(-1) and (1.1 +/- 0.2) x 10(5) M(-1) s(-1) (pH 7, 25 degrees C)). Significant differences were observed in the H2O2-mediated conversion of compound II to compound III as well as in the spectral features of compound II. When compared with HRP and other heme peroxidases, in Y249F, this reaction is significantly faster ((1.2 +/- 0.2) x 10(4) M(-1) s(-1))). Ferrous wild-type KatG was also rapidly converted by hydrogen peroxide in a two-phasic reaction via compound II to compound III (approximately 2.0 x 10(5) M(-1) s(-1)), the latter being also efficiently transformed to ferric KatG. These findings are discussed with respect to a proposed mechanism for the catalatic activity.