Preparation of isomaltose oligosaccharides labelled with 14C in the non-reducing terminal unit, and their use in studies of dextranase activity

A series of end-labelled isomaltose oligosaccharides was prepared by the reaction of dextran-sucrase with sucrose-¹⁴C in the presence of excess of unlabelled isomaltose saccharides as alternative acceptor. The main product of each reaction contained one more D-glucose residue than the acceptor substrate, and the label was located at the non-reducing end. The end-labelled saccharides were used to determine the specificity of a bacterial dextranase that required five or more consecutive α-(1→6)-D-glucosidic linkages in the substrate. The third linkage from the reducing end of isomaltohexaose (IM₆) and of other substrates with longer chains (IM₇ and IM₈) was the most susceptible to attack, and the products from higher oligosaccharides were IM₃, IM₄, and IM₅. Isomaltopentaose (IM₅) was further hydrolysed to IM₃ and IM₂ when a 35-fold excess of enzyme was added, but there was no action on IM₄, IM₃, or IM₂ under these conditions. It was concluded that the dextranase hydrolysed linkages penultimate to either end of the chain only with difficulty, and that end linkages were completely resistant to attack.     

STUDIES IN THE PHYSIOLOGY OF FUSARIA. THE RESPIRATORY AND FERMENTATIVE MECHANISMS

1.Fusarium tricothecoides was selected for a study of the respiratory and fermentative activities of Fusaria. "Resting cell" suspensions were investigated by the Barcroft manometric technique. 2. The results of the investigation indicate clearly that the mechanism of endogenous metabolism (respiration) is distinct from the exogenous mechanism (fermentation). Anaerobically no significant CO₂ production is apparent without added substrate. In the presence of glucose the anaerobic CO₂ evolution is practically equal to the added CO₂ evolved aerobically in the presence of added glucose. Low concentrations of iodoacetate or fluoride selectively poison the exogenous mechanism but do not affect the endogenous mechanism. Alcohol is not produced in the course of endogenous metabolism, but is produced in the presence of added glucose. 3. A study of the metabolism of the organism throughout its entire growth phase from 1 to 7 days has been made. 4. The ability of suspensions of Fusarium sp. H., obtained by growth on a variety of common substrates, to attack a large number of carbon sources with the production of exogenous CO₂ was determined. It is found that organisms grown on glucose will attack only glucose, mannose, and fructose, but none of the common intermediary metabolites except pyruvic acid. Organisms grown on galactose attack galactose, as well as the other hexoses, indicating an adaptive mechanism. 5. An identical mechanism for the dissimilation of glucose, mannose, and galactose is indicated since no additive effects with these substrates were observed. Growths on non-hexose carbon sources attack glucose slightly under the experimental conditions with the evolution of CO₂, but do not attack any other substrate. This would indicate a residual glucose-dissimilating mechanism in all growths investigated. 6. Striking similarities between the general metabolism of resting suspensions of Fusarium sp. H. and resting suspensions of yeast cells are apparent.     

Isolation and Characterization of Two Geometric Allene Oxide Isomers Synthesized from 9S-Hydroperoxylinoleic Acid by Cytochrome P450 CYP74C3: STEREOCHEMICAL ASSIGNMENT OF NATURAL FATTY ACID ALLENE OXIDES*

Specialized cytochromes P450 or catalase-related hemoproteins transform fatty acid hydroperoxides to allene oxides, highly reactive epoxides leading to cyclopentenones and other products. The stereochemistry of the natural allene oxides is incompletely defined, as are the structural features required for their cyclization. We investigated the transformation of 9S-hydroperoxylinoleic acid with the allene oxide synthase CYP74C3, a reported reaction that unexpectedly produces an allene oxide-derived cyclopentenone. Using biphasic reaction conditions at 0 °C, we isolated the initial products and separated two allene oxide isomers by HPLC at −15 °C. One matched previously described allene oxides in its UV spectrum (λ_max 236 nm) and NMR spectrum (defining a 9,10-epoxy-octadec-10,12Z-dienoate). The second was a novel stereoisomer (UV λ_max 239 nm) with distinctive NMR chemical shifts. Comparison of NOE interactions of the epoxy proton at C9 in the two allene oxides (and the equivalent NOE experiment in 12,13-epoxy allene oxides) allowed assignment at the isomeric C10 epoxy-ene carbon as Z in the new isomer and the E configuration in all previously characterized allene oxides. The novel 10Z isomer spontaneously formed a cis-cyclopentenone at room temperature in hexane. These results explain the origin of the cyclopentenone, provide insights into the mechanisms of allene oxide cyclization, and define the double bond geometry in naturally occurring allene oxides.     

Influence of water activity on the enzyme biosynthesis and enzyme activities produced by Trichoderma viride TS in solid-state fermentation

Influence of water activity (a_w) on biosynthesis of polygalacturonase, d-xylanase and β-glucosidase in solid culture system of Trichoderma viride TS was studied. It was found that the production of enzymes was strongly affected by water activity of substrate and nature of a_w depressor used. The polygalacturonase and d-xylanase production were maximized at a_w = 0.995 whereas β-glucosidase formation was favored at a_w = 0.96–0.98. The influence of water activity on catalytic effect of enzymes using sodium chloride, glycerol and sorbitol as a_w depressor was also investigated. It was observed that sorbitol improved the thermal stability of polygalacturonase and d-xylanase.     

In crystallo capture of a Michaelis complex and product-binding modes of a bacterial phosphotriesterase

The mechanism by which the binuclear metallophosphotriesterases (PTEs, E.C. 3.1.8.1) catalyse substrate hydrolysis has been extensively studied. The μ-hydroxo bridge between the metal ions has been proposed to be the initiating nucleophile in the hydrolytic reaction. In contrast, analysis of some biomimetic systems has indicated that μ-hydroxo bridges are often not themselves nucleophiles, but act as general bases for freely exchangeable nucleophilic water molecules. Herein, we present crystallographic analyses of a bacterial PTE from Agrobacterium radiobacter, OpdA, capturing the enzyme-substrate complex during hydrolysis. This model of the Michaelis complex suggests the alignment of the substrate will favour attack from a solvent molecule terminally coordinated to the α-metal ion. The bridging of both metal ions by the product, without disruption of the μ-hydroxo bridge, is also consistent with nucleophilic attack occurring from the terminal position. When phosphodiesters are soaked into crystals of OpdA, they coordinate bidentately to the β-metal ion, displacing the μ-hydroxo bridge. Thus, alternative product-binding modes exist for the PTEs, and it is the bridging mode that appears to result from phosphotriester hydrolysis. Kinetic analysis of the PTE and promiscuous phosphodiesterase activities confirms that the presence of a μ-hydroxo bridge during phosphotriester hydrolysis is correlated with a lower pK_a for the nucleophile, consistent with a general base function during catalysis.     

Reductive Reactivity of Iron(III) Oxides in the East China Sea Sediments: Characterization by Selective Extraction and Kinetic Dissolution

Reactive Fe(III) oxides in gravity-core sediments collected from the East China Sea inner shelf were quantified by using three selective extractions (acidic hydroxylamine, acidic oxalate, bicarbonate-citrate buffered sodium dithionite). Also the reactivity of Fe(III) oxides in the sediments was characterized by kinetic dissolution using ascorbic acid as reductant at pH 3.0 and 7.5 in combination with the reactive continuum model. Three parameters derived from the kinetic method: m ₀ (theoretical initial amount of ascorbate-reducible Fe(III) oxides), k′ (rate constant) and γ (heterogeneity of reactivity), enable a quantitative characterization of Fe(III) oxide reactivity in a standardized way. Amorphous Fe(III) oxides quantified by acidic hydroxylamine extraction were quickly consumed in the uppermost layer during early diagenesis but were not depleted over the upper 100 cm depth. The total amounts of amorphous and poorly crystalline Fe(III) oxides are highly available for efficient buffering of dissolved sulfide. As indicated by the m ₀, k′ and γ, the surface sediments always have the maximum content, reactivity and heterogeneity of reactive Fe(III) oxides, while the three parameters simultaneously downcore decrease, much more quickly in the upper layer than at depth. Albeit being within a small range (within one order of magnitude) of the initial rates among sediments at different depths, incongruent dissolution could result in huge discrepancies of the later dissolution rates due to differentiating heterogeneity, which cannot be revealed by selective extraction. A strong linear correlation of the m ₀ at pH 3.0 with the dithionite-extractable Fe(III) suggests that the m ₀ may represent Fe(III) oxide assemblages spanning amorphous and crystalline Fe(III) oxides. Maximum microbially available Fe(III) predicted by the m ₀ at pH 7.5 may include both amorphous and a fraction of other less reactive Fe(III) phases.     

Manganese Reduction by Microbes from Oxic Regions of the Lake Vanda (Antarctica) Water Column

Depth profiles of metals in Lake Vanda, a permanently ice-covered, stratified Antarctic lake, suggest the importance of particulate manganese oxides in the scavenging, transport, and release of metals. Since manganese oxides can be solubilized by manganese-reducing bacteria, microbially mediated manganese reduction was investigated in Lake Vanda. Microbes concentrated from oxic regions of the water column, encompassing a peak of soluble manganese [Mn(II)], reduced synthetic manganese oxides (MnO₂) when incubated aerobically. Pure cultures of manganese-reducing bacteria were readily isolated from waters collected near the oxic Mn(II) peak. Based on phylogenetic analysis of the 16S rRNA gene sequence, most of the isolated manganese reducers belong to the genus Carnobacterium. Cultures of a phylogenetically representative strain of Carnobacterium reduced synthetic MnO₂ in the presence of sodium azide, as was seen in field assays. Unlike anaerobes that utilize manganese oxides as terminal electron acceptors in respiration, isolates of the genus Carnobacterium reduced Mn(IV) via a diffusible compound under oxic conditions. The release of adsorbed trace metals accompanying the solubilization of manganese oxides may provide populations of Carnobacterium with a source of nutrients in this extremely oligotrophic environment.     

Altering the Substrate Specificity of Organophosphorus Hydrolase for Enhanced Hydrolysis of Chlorpyrifos

Chlorpyrifos is one of the most popular pesticides used for agriculture crop protection, and widespread contamination is a potential concern. However, chlorpyrifos is hydrolyzed almost 1,000-fold slower than the preferred substrate, paraoxon, by organophosphorus hydrolase (OPH), an enzyme that can degrade a broad range of organophosphate pesticides. We have recently demonstrated that directed evolution can be used to generate OPH variants with up to 25-fold improvement in hydrolysis of methyl parathion. The obvious question and challenge are whether similar success could be achieved with this poorly hydrolyzed substrate, chlorpyrifos. For this study, five improved variants were selected from two rounds of directed evolution based on the formation of clear haloes on Luria-Bertani plates overlaid with chlorpyrifos. One variant, B3561, exhibited a 725-fold increase in the k_cat/K_m value for chlorpyrifos hydrolysis as well as enhanced hydrolysis rates for several other OP compounds tested. Considering that wild-type OPH hydrolyzes paraoxon at a rate close to the diffusion control limit, the 39-fold improvement in hydrolysis of paraoxon by B3561 suggests that this variant is one of the most efficient enzymes available to attack a wide spectrum of organophosphate nerve agents.     

Efficient preparation of enantiopure l-tert-leucine through immobilized penicillin G acylase catalyzed kinetic resolution in aqueous medium

Racemic _DL-tert-leucine (_DL-Tle) was resolved to obtain enantiopure _L-Tle through enantioselective hydrolysis of its N-phenylacetyl derivative with immobilized penicillin G acylase (PGA). The effects of pH, reaction temperature, substrate concentration and reaction time on the reaction were investigated. The reaction was conveniently carried out at 0.4 M substrate concentration in water at pH 8.0 and 30 °C. Under the optimized reaction conditions, _L-Tle was obtained in an enantiopure form (>99% ee) with 45.8% substrate conversion after 4 h. The thermal stability and operational stability of immobilized PGA were examined. Furthermore, the preparation of _L-Tle was successfully performed in a recirculating packed bed reactor (RPBR) system and immobilized PGA exhibited a long-term stability for 51 days with a slight decrease of activity. The isolated _D-enantiomer was racemized at 160 °C for 15 min and reused as substrate. The results obtained clearly demonstrated a potential for industrial application of immobilized PGA in the preparation of _L-Tle through enantioselective hydrolysis of its N-phenylacetyl derivative.     

Purification and specificity of RNase A3 from Vicia faba root cells

Vicia faba root ribonucleases are bound to Cibacron blue F3GA. A Blue dextran-Sepharose column was used to purify RNase A₃, the more abundant enzyme from V. faba root. Using dinucleoside monophosphate as substrates, it appears that this enzyme behaves as a cyclizing phosphotransferase. With high enzyme/substrate ratios on prolonged digestion a partial release of a nucleoside 3′ phosphate occurs. The specificity is relatively high since only the purine-purine phosphodiester linkages out of 16 types of possible links are easily cleaved. When a pyrimidine is involved in the phosphodiester bond, a much slower rate of attack (Py in 5′) or no attack (Py in 3′) was detected.     

Stereoselectivity in deacylation of nitrophenyl acylates by poly(ethylenimine) derivatives

Deacylation of nitrophenyl acetates containing carboxyl substituents [4-acetoxy-3-nitrobenzoic acid (1), 3-acetoxy-4-nitrobenzoic acid (2), and 2-acetoxy-5-nitrobenzoic acid (3)] was studied in the presence of poly(ethylenimine) derivatives. The polymers examined contained lauryl groups (Lau₁₂PEI) or both lauryl and imidazolyl groups (Lau₁₂Im₁₀PEI). The reaction with active ester proceeds through the attack of primary amino groups of the polymers at the acyl carbons of the substrates. The reaction of Lau₁₂Im₁₀PEI with a hydrophobic ester, p-nitrophenyl caproate (NPC), however, has been reported to involve the attack by the imidazolyl group of the polymer. Thus, the anionic (carboxyl-containing) and the hydrophobic esters bind to different domains on Lau₁₂Im₁₀PEI. Among the anionic substrates, 3 has uniquely large k_cat values compared with 1 or 2. This is explained in terms of closer proximity between a nucleophile amino group of the polymer and the scissile bond of the substrate in the polymer-substrate complex.     

Purification and Properties of an S-Adenosylmethionine: 2,4-Disubstituted Phenol O-Methyltransferase from Phanerochaete chrysosporium

An enzyme catalyzing the O-methylation of acetovanillone (3-methoxy-4-hydroxyacetophenone) by S-adeno-sylmethionine was isolated from Phanerochaete chrysosporium and purified 270-fold by ultrafiltration, anion-exchange chromatography, and gel filtration. The enzyme exhibited a pH optimum between 7 and 9 and was rapidly denatured at temperatures above 55°C. The K_m values for acetovanillone and S-adenosylmethionine were 34 and 99 μM, respectively. S-Adenosylhomocysteine acted as a powerful competitive inhibitor of S-adenosylmethionine, with a K_i of 41 μM. The enzyme was also susceptible to inhibition by thiol reagents and low concentrations of heavy metal ions. Gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the enzyme was monomeric and had a molecular weight of approximately 53,000. Substrate specificity studies showed that 3-methoxy- and 3,5-dimethoxy-substituted 4-hydroxy-benzaldehydes, -benzoic acids, and -acetophenones were the preferred substrates for the enzyme. The corresponding 3,4-dihydroxy compounds were methylated relatively slowly, while the 3-hydroxy-4-methoxy compounds were almost inactive as substrates. Substituents in both the 2 and 4 positions relative to the hydroxyl group appeared to be essential for significant enzyme attack of a substrate. Provided that certain steric criteria were satisfied, the nature of the substituent was not critical. Hence, xenobiotic compounds such as 2,4-dichlorophenol and 2,4-dibromophenol were methylated almost as readily as acetovanillone. However, an extended side chain in the 4 position was not compatible with activity as a substrate, and neither homovanillic, caffeic, nor ferulic acid was methylated. The substrate range of the O-methyltransferase tends to imply a role in the catabolism or detoxification of lignin degradation products such as vanillic and syringic acids.     

Transition state stabilization by deaminases: Rates of nonenzymatic hydrolysis of adenosine and cytidine

Nonenzymatic rates of hydrolytic deamination of adenosine and cytidine by acids and bases analogous to side chains of naturally occurring amino acids are compared with the rates of uncatalyzed deamination in water and with the rates of the hydroxide- and hydrogen ion-catalyzed reactions. For adenosine, hydroxide ion is an effective catalyst, with a second-order rate constant of 7.5 × 10⁻⁶ m⁻¹ s⁻¹ at 85°C and an energy of activation of 19.9 kcal/mol. Acid-catalyzed deamination of adenine proceeds with a second-order rate constant of 1.5 × 10⁻⁶ m⁻¹ s⁻¹ at 85°C. At concentrations of 1 m and at pH values corresponding to their respective pK_a values, dimethylamine, acetate, selenide, imidazole, phosphate, and zinc(II) do not enhance the rate of deamination of adenosine beyond that observed in water, and 2-mercaptoethanol produces only a modest rate enhancement. The uncatalyzed rate of adenosine deamination in water is 8.6 × 10⁻⁹ s⁻¹ at 85°C: extrapolation to 37°C and comparison with k_cat for rat hepatoma adenosine deaminase yield a rate enhancement by the enzyme of approximately 2 × 10¹²-fold. 1,6-Dimethyladenosine, the conjugate acid of which has a pK_a value much higher than that of adenosine, is not readily deaminated, suggesting that the uncatalyzed deamination of adenosine does not proceed by hydroxide ion attack on the rare protonated form of adenosine, but rather by attack on the neutral species. Deamination of cytidine is catalyzed most effectively by hydroxide ion, with a second-order rate constant of 4.5 × 10⁻⁴ m⁻¹ s⁻¹ at 85°C and an energy of activation of 28.5 kcal/mol. The uncatalyzed rate of deamination of cytidine in water, which also exhibits an energy of activation of 28.5 kcal/mol, is 8.8 × 10⁻⁸ s⁻¹ at 85°C. Comparison of the rate extrapolated to 25°C with k_cat for bacterial cytidine deaminase gives a rate enhancement for the enzyme of 4 × 10¹¹-fold. The C-5 proton of the pyrimidine ring of cytidine does not exchange with solvent during alkaline hydrolysis, suggesting that deamination under these conditions does not involve prior addition of water across the 5,6 double bond.     

The hydrolysis of 3-(2-furylacryloyl)-glycyl-l-leucine amide by thermolysin

The kinetics of the hydrolysis of 3-(2-furylacryloyl)-glycycl-l-leucine amide by thermolysin has been reinvestigated. It was found that the K_m for the enzyme substrate interaction is 2.5 × 10^?3m at pH 7.2. This K_m is an order of magnitude less than what has been previously assumed to be the K_m for the enzyme-substrate interaction. The normally recommended assay has 1–3 × 10^?3m substrate and is based on the assumption that the substrate concentration is much less than the K_m. Our data indicate that this assumption appears to be invalid. The hydrolysis of 3-(2-furylacryloyl)-glycyl-l-leucine amide results in a maximum decrease in absorbance at 322 nm. The change in absorbance is nearly 10-fold greater at 322 nm than the change in absorbance at 345 nm where the hydrolysis has been customarily followed. By following the hydrolysis of the substrate at 10^?4m at 322 nm it is possible to work under conditions where the substrate concentration is much less than the K_m.     

The enzymatic mechanism of carboxypeptidase: A molecular dynamics study

An MD simulation of the system carboxypeptidase A (CPA) with the tetrapeptide Val-Leu-Phe-Phe has been performed in order to learn about the substrate disposition just prior to nucleophilic attack. We have explored the model in which the substrate does not substitute the zinc-coordinated water (the “water” mechanism). The simulations do suggest as feasible that the Zn-OH₂ group performs a nucleophilic attack on the Phe-Phe peptidic bond. We have also investigated the model in which the carbonyl oxygen displaces the zinc-coordinated water. In this case the substrate and Glu-270 orient themselves to allow an anhydride intermediate during the peptidic bond cleavage (the “anhydride” mechanism). Based on the results of the simulations, both “water” and “anhydride” mechanisms are structurally feasible, although the former model seems more probable on chemical grounds. © 1994 John Wiley & Sons, Inc.     

The Hydrogenase Activity of the Molybdenum/Copper-containing Carbon Monoxide Dehydrogenase of Oligotropha carboxidovorans

The reaction of the air-tolerant CO dehydrogenase from Oligotropha carboxidovorans with H₂ has been examined. Like the Ni-Fe CO dehydrogenase, the enzyme can be reduced by H₂ with a limiting rate constant of 5.3 s⁻¹ and a dissociation constant K_d of 525 μm; both k_red and k_red/K_d, reflecting the breakdown of the Michaelis complex and the reaction of free enzyme with free substrate in the low [S] regime, respectively, are largely pH-independent. During the reaction with H₂, a new EPR signal arising from the Mo/Cu-containing active site of the enzyme is observed which is distinct from the signal seen when the enzyme is reduced by CO, with greater g anisotropy and larger hyperfine coupling to the active site ^63,65Cu. The signal also exhibits hyperfine coupling to at least two solvent-exchangeable protons of bound substrate that are rapidly exchanged with solvent. Proton coupling is also evident in the EPR signal seen with the dithionite-reduced native enzyme, and this coupling is lost in the presence of bicarbonate. We attribute the coupled protons in the dithionite-reduced enzyme to coordinated water at the copper site in the native enzyme and conclude that bicarbonate is able to displace this water from the copper coordination sphere. On the basis of our results, a mechanism for H₂ oxidation is proposed which involves initial binding of H₂ to the copper of the binuclear center, displacing the bound water, followed by sequential deprotonation through a copper-hydride intermediate to reduce the binuclear center.     

Rhizome architecture inPhragmites australis in relation to water depth: Implications for within-plant oxygen transport distances

Phragmites australis (Cav.)Trin. exSteud. is a perennial plant, largely relying on its rhizomes for resource storage, spreading and anchorage in the substrate. Vertical distribution and length of horizontal rhizomes ofPhragmites australis were investigated at the reed bed edge in a lake in southern Sweden. In deep water, horizontal rhizomes were relatively short and superficially situated in the substrate. It is hypothesised that this is an adaptation to water depth by keeping O₂-transport distances through shoots and rhizomes as short as possible. In shallow water,P. australis rhizomes generally penetrated deeply into the substrate, probably improving anchorage and nutrient uptake possibilities. Further, horizontal rhizomes were longer in shallow water, which may increase the rate of vegetative spread. Because of these changes in rhizome architecture, “critical within-plant oxygen transport distances” did not change with water depth. This indicates thatP. australis maximises the extension of its rhizomes in relation to spatial differences in water depth. This may limit the ability ofP. australis to tolerate sudden temporal increases in water depth or eutrophication.     

A different approach to generating the data for parameter estimation with the heterotrophic activity method

This paper outlines a different approach to generating the data for V_max and T_t estimation with the Wright-Hobbie [1] method of measuring heterotrophic activities in aquatic environments. To be certain that the incubation times chosen are appropriate for all concentrations of substrate tested, and to increase the precision of the kinetic parameter estimates, we have adopted the approach of using kinetic plots derived from independent time-course studies performed at each concentration of substrate and analyzed by non-linear regression analysis. In keeping with our interest in the impact of acidification on aquatic microbial activities, we have applied this approach to the sediments and water column of the acid-stressed Silver Lake.     

Probing mechanism of metal catalyzed hydrolysis of Thymidylyl (3′-O, 5′-S) thymidine phosphodiester derivatives

Hydrolysis of nucleic acids is of fundamental importance in biological sciences. Kinetic and theoretical studies on different substrates wherein the phosphodiester bond combined with alkyl or aryl groups and sugar moiety have been the focus of attention in recent literature. The present work focuses on understanding the mechanism and energetics of alkali metal (Li, Na, and K) catalyzed hydrolysis of phosphodiester bond in modeled substrates including Thymidylyl (3′-O, 5′-S) thymidine phosphodiester (Tp-ST) (1), 3′-Thymidylyl (1-trifluoroethyl) phosphodiester (Tp-OCH₂CF₃) (2), 3′-Thymidylyl (o-cholorophenyl) phosphodiester (Tp-OPh(o-Cl)) (3) and 3′-Thymidylyl(p-nitrophenyl) phosphodiester (Tp-OPh(p-NO₂)) (4) employing density functional theory. Theoretical calculations reveal that the reaction follows a single-step (A_ND_N) mechanism where nucleophile attack and leaving group departure take place simultaneously. Activation barrier for potassium catalyzed Tp-ST hydrolysis (12.0 kcal mol^?1) has been nearly twice as large compared to that for hydrolysis incorporating lithium or sodium. Effect of solvent (water) on activation energies has further been analyzed by adding a water molecule to each metal ion of the substrate. It has been shown that activation barrier of phosphodiester hydrolysis correlates well with basicity of leaving group.

Characterization of recombinant human renin: Kinetics,pH-stability,and peptidomimetic inhibitor binding

The kinetic behavior andpH-stability of recombinant human renin was analyzed using a new fluorogenic substrate based on the normal P₆-P_3′ renin cleavage sequence in human angiotensinogen. The design of this fluorogenic substrate makes possible, for the first time, direct monitoring of the kinetics of proteolytic conversion of prorenin to renin. ThepH-stability profile for renin, measured with the substrate at 25°C, indicated a broad plateau of stability betweenpH 6.0 and 10.0. Analysis of thepH-activity profile of renin for the substrate indicated a minimumK _m (～1.8 µM) atpH ～7.4 and a maximumV _m betweenpH 7.4 and 8.0. The thermodynamics of the binding of a novel, soluble, peptidomimetic inhibitor to renin indicated it is possible to retain the tight-binding characteristics and enthalpy contributions to binding of larger peptide-derived inhibitors, while reducing inhibitor size and entropic contributions to binding. A novel derivative of the fluorogenic substrate, containing a 3-methyl histidine substitution at the P₂ site, was used to test the recent hypothesis that renin functions by virtue of substrate-directed catalysis.