Chlorophyllase in citrus leaves. Kinetic aspects of the reaction

The reaction: Chlorophyll + Enzyme → Chlorophyllide + Phytol follows a first order kinetics with regard to the quantity of enzyme, when it is saturated by substrate. K_m and V_m were determined from the average reaction rates for the substrates: Chlorophylla andb, pheophytina andb, methylchlorophyllidea and methylpheophorbide a: The lowest K_m corresponded to chlorophylla and the highest V_m to methylpheophorbidea. For a substrate of chlorophyll (a + b), K_m and V_m were determined also using the initial reaction rates. “Enzyme efficiency” was calculated using both methods of determination. The reaction products partially inhibit the hydrolytic process.     

Stereochemical control of yeast reductions. 2. Quantitative treatment of the kinetics of competing enzyme systems for a single substrate

Quantitative expressions have been developed for systems such as yeast reductions where competing enzymes act on one substrate to yield two enantiomeric products. These expressions relate the observed stereochemical variables, the extent of conversion (C), the optical purity expressed as enantiomeric excess (ee), and the initial substrate concentration (A₀) to the kinetic parameters K_R and K_S (apparent Michaelis constants) and y ($\text{V}{}_{\mathrm{R}}\text{V}{}_{\mathrm{S}}$, the ratio of maximal velocities) of such competing enzymes. The expressions have been experimentally verified using a purified competing enzyme system of l- and d-lactic dehydrogenases. Furthermore, the enantioselective reduction of β-keto esters by intact yeast cells has been examined by means of this kinetic analysis.     

Ordered substrate binding and evidence for a thermally induced change in mechanism for E. coli aspartate transcarbamylase

Isotopic exchange kinetics at equilibrium for E. coli native aspartate transcarbamylase at pH 7.8, 30 °C, are consistent with an ordered BiBi substrate binding mechanism. Carbamyl phosphate binds before l-Asp, and carbamyl-aspartate is released before inorganic phosphate. The rate of [¹⁴C]Asp C-Asp exchange is much faster than [³²P]carbamyl phosphate P_i exchange. Phosphate, and perhaps carbamyl phosphate, appears to bind at a separate modifier site and prevent dissociation of active-site bound P_i or carbamyl phosphate. Initial velocity studies in the range of 0–40 °C reveal a biphasic Arrhenius plot for native enzyme: E_a (>15 °C) = 6.3 kcal/ mole and E_a (<15 °C) = 22.1 kcal/mole. Catalytic subunits show a monophasic plot with E_a ? 20.2 kcal/mole. This, with other data, suggests that with native enzyme a conformational change accompanying aspartate association contributes significantly to rate limitation at t > 15 °C, but that catalytic steps become definitively slower below 15 °C. Model kinetics are derived to show that this change in mechanism at low temperature can force an ordered substrate binding system to produce exchange-rate patterns consistent with a random binding system with all exchange rates equal. The nonlinear Arrhenius plot also has important consequences for current theories of catalytic and regulatory mechanisms for this enzyme.     

Effect of internal diffusion in heterogeneous enzyme systems: Evaluation of true kinetic parameters and substrate diffusivity

The effect of internal diffusion on the overall reaction rate in spherical particles and membranes containing immobilized enzymes has been investigated theoretically. Since they represent open systems, the MichaelisMenten kinetics is obeyed in the absence of diffusional effects at steady state even at high enzyme concentrations. When internal diffusion perturbs the reaction, the system can not be described any more by K_M and V_max? alone, but is conveniently characterized by the modulus. Assuming that only internal diffusion interferes with the enzyme reaction, the effect of the modulus on the overall rate of reaction is illustrated by the results of computer calculations. Plots of the overall reaction rate against the substrate concentration are hyperbolas at various moduli for both membranes and spherical particles and no sigmoidal curves are obtained with immobilized enzyme systems. Since the conventional plots of enzyme kinetics do not yield straight lines under such conditions, a graphical method is proposed to determine K_M and V_max? as well as the substrate diffusivity in the enzymic medium.     

Differentiation of various types of biological oxidation of nitrogen in organic compounds

The various types of nitrogen which occur in organic compounds and which are susceptible to biological oxidation are clearly divided into groups depending upon the pK_a, of the constituent nitrogen. The enzymatic processes which give rise to the N-oxidation products are reviewed by a consideration of species differences, age of animal, pH optima, influence of inducing agents, inhibitors and microsomal pretreatments, as well as the stereochemistry of the nitrogen atom.From the data collected, a concept is developed which suggests that all basic amines (group I) are oxidised by a flavine adenine nucleotide (FAD)-dependent enzyme system, whereas non-basic nitrogen-containing compounds (group III) are oxidised by a cytochrome P450-dependent system.It is further suggested that compounds of intermediary pK_a,i.e. between 1 and 7 (group II), may be substrates for both enzyme systems, which would yield the same products, but by different processes. The extent to which N-oxidation occurs in a species would therefore depend on the pK_a of the substrate and the amounts and ratio of the two enzymes present, a lower pK_a favouring oxidation by the cytochrome P450 system and a higher pK_a favouring oxidation by the FAD system.In a similar manner, it is suggested that the oxidation of aromatic heterocyclic amines depends upon the pK_a of the nitrogen, compounds having a low pK_a being preferentially metabolised by nitrogen oxidation.     

Investigation of the operational stabilities and kinetics of glucoamylase immobilized on alkylamine derivatives of titanium(IV)-activated porous inorganic supports

Glucoamylase (exo-1,4-α-d-glucosidase, EC 3.2.3.1) was coupled to several porous silica matrices by an improved metal-link/chelation process using alkylamine derivatives of titanium(IV)-activated supports. In order to select the titanium activation procedure which gave stable enzyme preparations, long-term stability tests were performed. The immobilized glucoamylase preparations, in which the carrier was activated to dryness with a 15% w/v TiCl₄ solution, displayed very stable behaviour, with half-lives of ～60 days. The optimum operating conditions were determined for these preparations. There are significant differences between the behaviour of the immobilized enzyme and the free enzyme. The apparent K_m increased on immobilization due to diffusional resistances. The pH optimum for the immobilized preparation showed a slight shift to acid pH relative to that of the soluble enzyme. Also, the optimum temperature descreased to 60°C after immobilization. In order to test Michaelis-Menten kinetics at high degrees of conversion, time-course analysis of soluble starch hydrolysis was performed. It was observed that simple Michaelis-Menten kinetics are not applicable to the free/immobilized glucoamylase-starch system at high degrees of conversion.     

Action of sulphite on the substrate kinetics of chloroplastic NADP-dependent glyceraldehyde-3-phosphate dehydrogenase

The kinetics of NADP-GPD from spinach chloroplasts are biphasic vs NADPH and PGA. Thus, two maximum velocities exist with an intermediary plateau and two K_m values. Activation by NADPH + DTT increases V_max of both sections, but does not change the substrate affinities. Sulphite reduces the maximum activities of both sections vs NADPH, however, it causes normal substrate kinetics vs PGA; even V_max is reduced. Sulphite, present only during the activation process, suppresses the enzyme form with the higher V_max. The kinetics vs NADH are also biphasic; the activity is strongly reduced by preincubation of the chloroplasts with NADH + DTT or at NADH concentrations > 0.4mM. Using NADH as cofactor, inverted peaks in the kinetics vs PGA occur; sulphite is active in a similar way as when NADPH is used as cofactor. The biphasic kinetics are discussed with respect to additional potential for regulation of enzyme activity according to illumination and NADPH concentrations respectively.     

Allosteric behavior of platelet myosin.

The allosteric properties of platelet actomyosin and myosin have been further studied. At pH 7.2, both exhibit sigmoid kinetics with at least two interacting ATP binding sites. At pH 8.9, the velocity versus substrate curve is shifted to the right and becomes more sigmoidal. In contrast, at pH 5.5, the enzyme appears to follow hyperbolic kinetics and the K_m is reduced. In the presence of 1.4 m urea, the sigmoidicity is lost and the enzyme obeys Michaelis-Menten kinetics. The effect of ADP on the ATPase activity was also investigated. ADP shows characteristics of a competitive inhibitor; it increases K_m (shifts sigmoid curve to the right) without affecting V. When the enzyme is desensitized by low pH (5.5) or urea (1.4 m), the allosteric interaction is abolished without impairing the catalytic activity and ADP is no longer inhibitory. These findings suggest that platelet myosin possesses two interacting sites and that ADP binds to the allosteric site which appears to be different from the catalytic site.     

Energy cost for the proper ionization of active site residues in 6-phosphogluconate dehydrogenase from T. brucei

The catalytic mechanism of 6-phosphogluconate dehydrogenase requires the inversion of a Lys/Glu couple from its natural ionization state. The pK_a of these residues in free and substrate bound enzymes has been determined measuring by ITC the proton release/uptake induced by substrate binding at different pH values. Wt 6-phosphogluconate dehydrogenase from Trypanosoma brucei and two active site enzyme mutants, K185H and E192Q were investigated. Substrate binding was accompanied by proton release and was dependent on the ionization of a group with pK_a 7.07 which was absent in the E192Q mutant. Kinetic data highlighted two pK_a, 7.17 and 9.64, in the enzyme–substrate complex, the latter being absent in the E192Q mutant, suggesting that the substrate binding shifts Glu192 pK_a from 7.07 to 9.64. A comparison of wt and E192Q mutant appears to show that the substrate binding shifts Lys185 pK_a from 9.9 to 7.17. By comparing differences in proton release and the binding enthalpy of wt and mutant enzymes, the enthalpic cost of the change in the protonation state of Lys185 and Glu192 was estimated at ≈ 6.1 kcal/mol. The change in protonation state of Lys185 and Glu192 has little effect on Gibbs free energy, 240–325 cal/mol. However proton balance evidences the dissociation of other group(s) that can be collectively described by a single pK_a shift from 9.1 to 7.54. This further change in ionization state of the enzyme causes an increase of free energy with a total cost of 1.2–2.3 kcal/mol to set the enzyme into a catalytically competent form.     

Effects of ionization of p-nitrocatechol sulfate on its behavior as a substrate for arylsulfatases

Evidence is presented showing that arylsulfatases reacting at acidic pH are more active towards p-nitrocatechol sulfate substrate, while the reverse is true for the enzymes having alkaline pH_max. An explanation of this phenomenon is suggested based on the ionization of p-nitrocatechol sulfate (pK_a = 6.4) and a much higher reactivity of sulfatases with the acidic form of the substrate.     

Properties of water-insoluble matrix-bound lactate dehydrogenase.

Lactate dehydrogenase enzyme was immobilized by binding to a cyanogen bromideactivated Sepharose 4B-200 in 0.1 m phosphate buffer, pH 8.5. The immobilized enzyme was found to have lower K_m values for its substrates. K_m values for pyruvate and lactate were 8 × 10 ^?5m and 4 × 10^?3m, respectively, an order of magnitude less than the value for the native (free) enzyme. Chicken heart (H₄) lactate dehydrogenase was found to lose nearly all its substrate inhibition characteristics as a result of immobilization. The covalently bound muscle-type subunits of lactate dehydrogenase showed more favorable interaction with the muscle type than with the heart type subunits. An increase in thermal and acid stability of the dogfish muscle (M₄) lactate dehydrogenase as well as a decrease in the percentage of inhibition of enzyme activity by rabbit antisera and in the complement fixation was observed as a result of immobilization. The changes in the properties of the enzyme as a result of immobilization may be attributable to hindrance produced by the insoluble matrix as well as conformational changes in the enzyme molecules.     

Development of a fluorescent substrate to measure hyaluronidase activity

A novel fluorescent substrate (termed FRET-HA) to quantitatively assess hyaluronidase activity was developed. Hyaluronan (HA), the major substrate for hyaluronidase, was dual labeled with fluorescein amine and rhodamine B amine. The fluorescein amine fluorescence signal was significantly quenched and the rhodamine B amine signal was significantly enhanced due to fluorescence resonance energy transfer (FRET). In the presence of bovine testes hyaluronidase, cleavage of HA disrupted FRET, resulting in a loss of the fluorescein amine quenching that was dependent on both enzyme concentration and time. Increase in the fluorescein amine signal could be conveniently monitored in both noncontinuous and continuous fashions. The K_m value for bovine testes hyaluronidase was determined using FRET-HA in a continuous fluorescent assay. Importantly, the estimated K_m value for bovine testes hyaluronidase using FRET-HA as the substrate was in excellent agreement with K_m values reported previously for this enzyme using native (i.e., unlabeled) HA. Therefore, FRET-HA is a reliable substrate for quantitatively assessing the HA/hyaluronidase molecular interaction. The simplicity, sensitivity, and versatility of the FRET-HA substrate suggest that it will have utility in a variety of assay platforms and should be a new tool for assessing hyaluronidase activity.     

The nature of anion inhibition of human red cell carbonic anhydrases.

We studied anionic inhibition of the reaction CO₂ + OH^?? HCO₃^? catalyzed by human red cell carbonic anhydrase B (I) and C (II), using iodide and cyanate. In the forward reaction with respect to CO₂ as the substrate, inhibition was mixed but favoring noncompetitive; the back reaction, with HCO₃^? as the substrate, yielded strict competitive kinetics. Mean inhibition constants, K_I, in the pH range 7.2–7.5 are: iodide, 0.5 mm for enzyme B and 16 mm for C; cyanate, 0.8 μm for B and 20 μm for C. When OH^? was considered as the substrate for the forward reaction, cyanate and chloride behaved as competitive inhibitors. The true inhibition constant (K_I⁰) for cyanate (calculated for infinitely low OH^?) is 0.4 μm for enzyme B and 4 μm for C. Apart from the difference in anion affinity and some 10-fold higher activity of C > B, the isozymes showed similar patterns of inhibition. Data agree with generally proposed mechanisms describing the active site as ZnH₂O with pK_a of about 7.     

Influences of nonuniform activity distributions on the apparent maximum reaction rate and apparent Michaelis constant of immobilized enzyme reactions

The influences of nonuniform activity distribution within a porous solid support on the apparent kinetic parameters, V_m^app and K_m^app, of immobilized enzyme reactions following the Michaelis-Menten kinetics were theoretically investigated. As the enzyme is distributed to the neighborhood of the external surface of the support, V_m^app and K_m^app approach their respective intrinsic values over a wide range of substrate concentration. There is a close relationship between the nonuniform distribution and internal diffusional resistance. Changes in these two factors provide similar effects on V_m^app and K_m^app. As long as the immobilized enzyme reaction follows Michaelis-Menten kinetics, the nonuniform activity distribution never makes K_m^app less than its intrinsic value.     

Optimization of reaction conditions during enzyme immobilization on soluble carboxyl-containing carriers

The kinetics of 1-ethyl-3-(3′-dimethylaminopropyl)-carbodiimide interaction with carboxylic groups in low and high molecular weight compounds have been investigated for choosing the optimum conditions for enzyme immobilization on carboxylic carriers. Optimum pH values depended on the pK_a of an acid. The ionic strength of the reaction mixture influenced the process of activation with carbodiimide and a decrease in reaction rate accompanied an increase in ionic strength. Optimum conditions for the coupling process with the use of carbodiimide activation are suggested. The process of fibrinolysin immobilization on the soluble copolymer of acrylic acid and acrylamide with carbodiimide as a coupling agent was also studied.     

Purification and in situ immobilization of lipase from of a mutant of Trichosporon laibacchii using aqueous two-phase systems

The crude intracellular lipase (cell homogenate) from Trichosporon laibacchii was subjected to partial purification by aqueous two-phase system (ATPS) and then in situ immobilization by directly adding diatomites as carrier to the top PEG-rich phase of ATPS. A partition study of lipase in the ATPS formed by polyethylene glycol–potassium phosphate has been performed. The influence of system parameters such as molecular weight of PEG, system phase composition and system pH on the partitioning behaviour of lipase was evaluated. The ATPS consisting of PEG 4000 (12%) and potassium phosphate (K₂HPO₄, 13%) resulted in partition of lipase to the PEG-rich phase with partition coefficient 7.61, activity recovery 80.4%, and purification factor of 5.84 at pH of 7.0 and 2.0% NaCl. Moreover, the in situ immobilization of lipase in PEG phase resulted in a highest immobilized lipase activity of 1114.6 U g^?1. The above results show that this novel lipase immobilization procedure which couples ATPS extract and enzyme immobilization is cost-effective as well as time-saving. It could be potentially useful technique for the purification and immobilization of lipase.     

Pyruvate kinase variants of the Alaskan king-crab. Evidence for a temperature-dependent interconversion between two forms having distinct and adaptive kinetic properties

1. Pyruvate kinase of Alaskan king-crab leg muscle exists in two kinetically distinct forms, each of which displays a different temperature-dependence in the K_m for phosphoenolpyruvate. 2. A `cold' variant of the enzyme has hyperbolic kinetics and exhibits a minimal K<sub>m</sub> for substrate at 5°. At physiological concentrations of phosphoenolpyruvate the `cold' enzyme is active only below 10°. A `warm' pyruvate kinase has a minimal K<sub>m</sub> for substrate at about 12°. This enzyme displays sigmoidal kinetics and is likely to be inactive, at physiological substrate concentrations, at temperatures below 9°. 3. The combined activities of these two pyruvate kinases yield highly temperature-independent rates of catalysis, at physiological substrate concentrations, over the range of habitat temperatures encountered by the organism, namely 4–12°. 4. The two variants of pyruvate kinase do not appear to be isoenzymes in the conventional sense. Electrophoretic and electrofocus analyses revealed only single peaks of activity. 5. The results suggest that the `warm' pyruvate kinase and the `cold' pyruvate kinase are formed by a temperature-dependent interconversion of one protein species. This interconversion has major adaptive significance: as the temperature is lowered the `warm' enzyme is converted into the `cold' enzyme; the opposite situation obtains when the temperature is raised. Temperature changes thus mimic the effects noted for fructose 1,6-diphosphate on certain mammalian pyruvate kinases.     

Activity of Pseudomonas cepacia lipase in organic media is greatly enhanced after immobilization on a polypropylene support

The purified lipase from Pseudomonas cepacia was used as free and immobilized enzyme preparation for hydrolysis of p-nitrophenyl palmitate (pNPP) and p-nitrophenyl acetate (pNPA) in organic media. The free enzyme was mixed with bovine serum albumin and lyophilized. Immobilization was on porous polypropylene. Conditions where diffusional limitations of the substrate were not limiting the reaction rate were defined. The specific activity of the lipase was greatly enhanced upon immobilization: 16.5- and 7.8-fold for pNPP and pNPA respectively. Both the free and immobilized lipases followed Michaelis–Menten kinetics in organic solvent despite the heterogeneity (solid/liquid) of the reaction mixture. For pNPP, the activation factor upon immobilization came mainly from a reduction in K _m, app while k _cat was increased for pNPA. Received: 30 January 1997 / Accepted: 14 February 1997     

Alkaline phosphatase of sea urchin embryos: Chromatographic and electrophoretic characterization.

A change in the molecular form of alkaline phosphatase in sea urchin embryos accompanies the marked increase in activity that occurs at gastrulation. On the basis of chromatographic and electrophoretic analyses, two major classes of alkaline phosphatase are identified: early enzyme, from unfertilized eggs to mesenchyme blastula, characterized by a major peak of activity, with a K_av of 0.123 on Sephadex G-200 columns, elution from DEAE-Sephadex columns by 0.5 M NaCl, and a migration value of 0.51 (relative to bromophenol blue) after electrophoresis in 7.5% polyacrylamide gels; late enzyme, from gastrula to plutei, characterized by a K_av of 0.137, elution from DEAE-Sephadex by 0.55–0.75 M NaCl, and a migration value of 0.56. By chromatographic and electrophoretic criteria the early enzyme appears to have a slightly greater molecular volume, lower net negative charge, and more heterogeneous composition than the late enzyme. Both enzyme preparations were maximally active at a pH 9.4–9.5. Enzyme from all stages appears to be predominantly associated with cell membranes. Extracting the enzyme by treatment with n-butanol, precipitating the enzyme from the dialyzed aqueous phase with ethyl alcohol, and chromatographing the alcohol preparation on columns of sieving and anion-exchanging media resulted in a substantial purification of the enzyme from all stages.     

Human liver acid phosphatases: Purification and properties of a low-molecular-weight isoenzyme

A low-molecular-weight human liver acid phosphatase was purified 2580-fold to homogenity by a procedure involving ammonium sulfate fractionation, acid treatment, and SP-Sephadex ion-exchange chromatography with ion-affinity elution. The purified enzyme contains a single polypeptide chain and has a molecular weight of 14,400 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amino acid composition of this enzyme (E) is reported. A pH dependence study using p-nitrophenyl phosphate as a substrate (S) revealed the effect of substrate ionization (pK_a 5.2) and the participation of a group in the ES complex having a pK_a value of 7.8. The enzyme is readily inactivated by sulfhydryl reagents such as heavy metal ions. Alkylation of the enzyme with iodoacetic acid and iodoacetamide causes complete inactivation of the enzyme and this inactivation is prevented by the presence of phosphate ion. The enzyme is also inactivated by treatment with diethyl pyrocarbonate; protection against this reagent is afforded by phosphate ion. The substrate specificity of this enzyme is unusual for an acid phosphatase. Of the many alkyl and aryl phosphomonoesters tested, the only possibly physiological substrate hydrolyzed by this enzyme was flavin mononucleotide, which exhibits a V which is 3-fold larger at pH 5.0 and 6-fold larger at pH 7.0 than that for p-nitrophenyl phosphate. However, the enzyme also catalyzes the hydrolysis of acetyl phosphate at pH 5.0 with a velocity eight times larger than that reported for an acyl phosphatase from human erythrocytes.