Thermodynamic analysis of enzyme catalysed reactions: new insights into the Michaelis-Menten equation

A simple thermodynamic analysis of the well-known Michaelis-Menten equation (MME) of enzyme catalysis is proposed that employs the chemical potential mu to follow the Gibbs free energy changes attending the formation of the enzyme-substrate complex and its turnover to the product. The main conclusion from the above analysis is that low values of the Michaelis constant KM and high values of the turnover number k(cat) are advantageous: this supports a simple algebraic analysis of the MME, although at variance with current thinking. Available data apparently support the above findings. It is argued that transition state stabilisation - rather than substrate distortion or proximity - is the key to enzyme catalysis.

Genomic organisation, activity and distribution analysis of the microbial putrescine oxidase degradation pathway

The catalytic action of putrescine specific amine oxidases acting in tandem with 4-aminobutyraldehyde dehydrogenase is explored as a degradative pathway in Rhodococcus opacus. By limiting the nitrogen source, increased catalytic activity was induced leading to a coordinated response in the oxidative deamination of putrescine to 4-aminobutyraldehyde and subsequent dehydrogenation to 4-aminobutyrate. Isolating the dehydrogenase by ion exchange chromatography and gel filtration revealed that the enzyme acts principally on linear aliphatic aldehydes possessing an amino moiety. Michaelis-Menten kinetic analysis delivered a Michaelis constant (KM=0.014mM) and maximum rate (Vmax=11.2μmol/min/mg) for the conversion of 4-aminobutyraldehyde to 4-aminobutyrate. The dehydrogenase identified by MALDI-TOF mass spectrometric analysis (E value=0.031, 23% coverage) belongs to a functionally related genomic cluster that includes the amine oxidase, suggesting their association in a directed cell response. Key regulatory, stress and transport encoding genes have been identified, along with candidate dehydrogenases and transaminases for the further conversion of 4-aminobutyrate to succinate. Genomic analysis has revealed highly similar metabolic gene clustering among members of Actinobacteria, providing insight into putrescine degradation notably among Micrococcaceae, Rhodococci and Corynebacterium by a pathway that was previously uncharacterised in bacteria.

Role of amine oxidase expression to maintain putrescine homeostasis in Rhodococcus opacus

While applications of amine oxidases are increasing, few have been characterised and our understanding of their biological role and strategies for bacteria exploitation are limited. By altering the nitrogen source (NH4Cl, putrescine and cadaverine (diamines) and butylamine (monoamine)) and concentration, we have identified a constitutive flavin dependent oxidase (EC 1.4.3.10) within Rhodococcus opacus. The activity of this oxidase can be increased by over two orders of magnitude in the presence of aliphatic diamines. In addition, the expression of a copper dependent diamine oxidase (EC 1.4.3.22) was observed at diamine concentrations>1mM or when cells were grown with butylamine, which acts to inhibit the flavin oxidase. A Michaelis-Menten kinetic treatment of the flavin oxidase delivered a Michaelis constant (KM)=190μM and maximum rate (kcat)=21.8s(-1) for the oxidative deamination of putrescine with a lower KM (=60μM) and comparable kcat (=18.2s(-1)) for the copper oxidase. MALDI-TOF and genomic analyses have indicated a metabolic clustering of functionally related genes. From a consideration of amine oxidase specificity and sequence homology, we propose a putrescine degradation pathway within Rhodococcus that utilises oxidases in tandem with subsequent dehydrogenase and transaminase enzymes. The implications of PUT homeostasis through the action of the two oxidases are discussed with respect to stressors, evolution and application in microbe-assisted phytoremediation or bio-augmentation.

Image -asparaginases from Mycobacterium tuberculosis strains H37Rv and H37Ra

M. tuberculosis H37Ra possesses two Image -asparaginases while the H37Rv strain possesses only a single enzyme. These enzymes have been purified and their properties studied. The two Image -asparaginases in H37Ra strain differ from each other in pH optima, heat inactivation, Michaelis constant and effects of inhibitors, while one of them resembles the single Image -asparaginase present in the H37Rv strain. Image -Cysteine inhibits both Image -asparaginases in an allosteric manner probably because it is one of the end-products in Image -asparagine metabolism. This is the first time that a qualitative difference has been reported in the enzyme pattern between the avirulent and virulent strains of M. tuberculosis.

Indoleacetaldoxime hydro-lyase : II. Purification and properties

The purification and some properties of the enzyme indoleacetaldoxime hydrolyase (EC 4.2.1.29) from the fungus Gibberella fujikuroi, which dehydrates indoleacetaldoxime (IAOX) to indoleacetonitrile (IAN), are described. The enzyme activity in the fungus is present only under certain culture conditions. It is a soluble enzyme, has an optimum pH at 7, shows an energy of activation of —15,670 cal/mole, and has a Michaelis constant of 1.7 × 10−4 Image at 30 °. It appears to be specific for IAOX, and 1 mole of IAN is produced per mole of IAOX utilized. The enzyme is inhibited by a number of aldoximes of which phenylacetaldoxime (PAOX) is the most potent inhibitor. Inhibition by PAOX is competitive (Ki = 2.2 × 10−8 Image ). The enzyme is inhibited by SH reagents such as p-hydroxymercuribenzoate and N-ethylmaleimide, and by a number of SH compounds such as cysteine, β-mercaptoethanol, and 2,3-dimercaptopropanol (BAL). However, glutathione activates the enzyme. Metal chelating agents such as 8-OH-quinoline and diethyl dithiocarbamate inhibit the enzyme; the inhibition is partly reversed by ferric citrate. Ascorbic acid, and particularly dehydroascorbic acid (DHA), are good activators of the enzyme. Several other biological oxidants had either no action or had a slight effect. Potassium cyanide activates the enzyme at low concentration but inhibits at higher concentrations. Reduction of the enzyme with NaBH4 reduces activity, and the effect is partly reversed by pyridoxal phosphate and also by DHA. The above properties indicate that both an SH function and an oxidized function are required for activity.

Purification and characterization of endo-1,4-β-xylanase from Paecilomyces varioti Bainier

An endo-xylanase (1,4-β-d-xylanxylanohydrolase EC 3.2.1.8) was isolated from the culture filtrate of Paecilomyces varioti Bainier. The enzyme was purified 3.2 fold with a 60% yield by gel filtration and ion exchange chromatography. The purified enzyme had a molecular weight of 25,000 with a sedimentation coefficient of 2.2 S. The isoelectric point of the enzyme was 3.9. The enzyme was obtained in crystalline form. The optimum pH range was 5.5–7.0 and the temperature, 65°C. The Michaelis constant was 2.5 mg larchwood xylan/ml. The enzyme was found to degrade xylan by an endo mechanism producing arabinose, xylobiose, xylo- and arabinosylxylo-oligosaccharides, during the initial stages of hydrolysis. On prolonged incubation, xylotriose, arabinosylxylotriose and xylobiose were the major products with traces of xylotetraose, xylose and arabinose.

Temperature response of trehalase from a mesophilic (Neurospora crassa) and a thermophilic (Thermomyces lanuginosus) fungus

The purified trehalases of the mesophilic fungus, Neurospora crassa, and the thermophilic fungus, Thermomyces lanuginosus, had similar temperature and pH optima for activity, but differed in molecular weight, electrophoretic mobility and Michaelis constant. At lower concentration, trehalases from both fungi were inactivated to similar extent at 60°C. While purified trehalase of T. lanuginosus was afforded protection against heat-inactivation by proteinaceous protective factor(s) present in mycelial extracts, by bovine serum albumin and by casein, these did not afford protection to N. crassa trehalase against heat inactivation. Both trehalases exhibited discontinuous Arrhenius plots with temperature of discontinuity at 40°C. The activation energy calculated from the slope of the Arrhenius plot was higher for the T. lanuginosus enzyme. The plots of apparent K m versus 1/T for trehalases of N. crassa and T. lanuginosus were linear from 30° to 60°C. The results show that purified trehalases of the mesophilic and the thermophilic fungus are distinct. Although, these exhibit similar thermostability of their catalytic function at low concentration, distinctive thermal stability characteristics of thermophilic enzyme become apparent at high protein concentration. This could be brought about in the cell by the enzyme itself, or by other proteins.

Actions of two serine proteases from Trimeresurus jerdonii venom on chromogenic substrates and fibrinogen

Jerdonobin and jerdofibrase are two serine proteases purified from the venom of Trimeresurus jerdonii. The Michaelis constant K-m and the catalytic rate constant K-cat of jerdonobin or jerdofibrase on three chromogenic substrates, H-D-Pro-Phe-Arg-pNA (S2302), H-D-Phe-pipecolyl-Arg-pNA (S2238), and H-D-Val-Leu-Lys-pNA (S2251) were obtained from lineweaver-Burk plots. Jerdofibrase could hydrolyze all three substrates, but jerdonobin had no detectable activity on S2251, suggesting a relatively broader substrate specificity for jerdofibrase than jerdonobin. By SDS-PAGE, jerdofibrase preferentially degraded Bbeta-chain of fibrinogen. It also degraded Aalpha-chain of fibrinogen with relatively slow activity, but did not act on the gamma-chain. In contrast, jerdonobin did not degrade fibrinogen within 12 h. Fibrinopeptides liberation test, identified by HPLC, showed jerdonobin released fibrinopeptide A and a small amount of fibrinopeptide B. Unlike jerdonobin, jerdofibrase mainly released fibrinopeptide B. These results indicate that the two enzymes differ in their ability to hydrolyze chromogenic substrates and in their actions on fibrinogen. (C) 2002 Elsevier Science Inc. All rights reserved.

Seasonal variation in kinetic parameters of alkaline phosphatase activity in a shallow Chinese freshwater lake (Donghu Lake)

Seasonal variation of the kinetic parameters of total alkaline phosphatase activity (APA) was studied in a shallow Chinese freshwater lake (Donghu Lake). At the three experimental stations the values of V-max of APA were higher and the negative correlation between orthophosphate and the total APA specific activity (V-max/Chl.) was stronger during summer (from June to September) P depletion. At the same time, the values of Michaelis constant (K-m) of APA at the three stations decreased. Phytoplankton seem to compensate for their phosphorus deficiency not only by an increase in enzyme production but also by an improved ability to use low substrate concentrations. (C) 1997 Elsevier Science Ltd.

Characterization of procaine metabolism as probe for the butyrylcholinesterase enzyme investigation by simultaneous determination of procaine and its metabolite using capillary electrophoresis with electrochemiluminescence detection

Capillary electrophoresis with electrochemiluminescene detection was used to characterize procaine hydrolysis as a probe for butyrylcholinesterase by in vitro procaine metabolism in plasma with butyrylcholinesterase acting as bioscavenger. Procaine and its metabolite N,N-diethylethanolamine were separated at 16 kV and then detected at 1.25 V in the presence of 5.0 mM Ru(bpy)(3)(2+), with the detection limits of 2.4 x 10(-7) and 2.0 x 10(-8) mol/L (S/N=3), respectively. The Michaelis constant K-m value was 1.73 x 10(-4) mol/L and the maximum velocity V-max was 1.62 x 10(-6) mol/L/min. Acetylcholine bromide and choline chloride presented inhibition effects on the enzymatic cleavage of procaine, with the 50% inhibition concentration (IC50) of 6.24 x 10(-3) and 2.94 x 10(-4) mol/L.

Kinetic study of paracetamol on prolidase activity in erythrocytes by capillary electrophoresis with Ru(bpy)(3)(2+) electrochemiluminescence detection

We explored the CE with Ru(bpy)(3)(2+) electrochemiluminescence detection for the kinetic study of drug-enzyme interaction. Effects of four nonsteroidal anti - inflammatory drugs including aspirin, paracetamol, sodium salicylate and phenacetin on prolidase (PLD) activity in erythrocytes were investigated. Aspirin enhanced PLD activity whereas the other three had inhibiting effects. This may reveal their different effects on the collagen biosynthesis and catabolism that influence tumor invasiveness. Kinetic study of paracetamol on PLD showed that the value of Michaelis constant Km for PLD was 1.23 mM. The mechanism of PLD inhibition by paracetamol is noncompetitive inhibition, and the inhibitor constant K-i value obtained in our research was 9.73 x 10(3) mu g/L.

Mechanistic studies on human N-acetylgalactosamine kinase

N-Acetylgalactosamine kinase (GALK2) is a small molecule kinase from the GHMP family which phosphorylates N-acetylgalactosamine at the expense of ATP. Recombinant GALK2 expressed in, and purified from, Escherichia coli was shown to be active with the following kinetic parameters: Michaelis constant for ATP, 14 +/- 3 mu M; Michaelis constant for N-acetylgalactosamine, 40 +/- 14 mu M; and turnover number, 1.0 +/- 0.1 s

Effects of alcohols and compatible solutes on the activity of β-galactosidase

During alcoholic fermentation, the products build up and can, ultimately, kill the organism due to their effects on the cell's macromolecular systems. The effects of alcohols on the steady-state kinetic parameters of the model enzyme ß-galactosidase were studied. At modest concentrations (0 to 2 M), there was little effect of methanol, ethanol, propanol and butanol on the kinetic constants. However, above these concentrations, each alcohol caused the maximal rate, V (max), to fall and the Michaelis constant, K (m), to rise. Except in the case of methanol, the chaotropicity of the solute, rather than its precise chemical structure, determined and can, therefore, be used to predict inhibitory activity. Compounds which act as compatible solutes (e.g. glycerol and other polyols) generally reduced enzyme activity in the absence of alcohols at the concentration tested (191 mM). In the case of the ethanol- or propanol-inhibited ß-galactosidase, the addition of compatible solutes was unable to restore the enzyme's kinetic parameters to their uninhibited levels; addition of chaotropic solutes such as urea tended to enhance the effects of these alcohols. It is possible that the compatible solutes caused excessive rigidification of the enzyme's structure, whereas the alcohols disrupt the tertiary and quaternary structure of the protein. From the point of view of protecting enzyme activity, it may be unwise to add compatible solutes in the early stages of industrial fermentations; however, there may be benefits as the alcohol concentration increases.

Hydration, conformational states and kinetics of yeast hexokinase PII /

The kinetic study of the coupled enzymatic reaction involving monomeric yeast hexokinase PII (HK) and yeast glucose-6-phosphate dehydrogenase (G-6-PDH) yields a Michaelis constant of 0.15 ± 0.01 mM for D-glucose. At pH 8.7 HK is present in monomeric form. The addition of polyethylene glycol (PEG), to the reaction mixture increased the affinity of HK for glucose, independent ofMW of the PEG from 2000 to 10000. The osmotic stress exerted by PEG can be used to measure the change in number of water molecules that accompany enzyme conformational changes (Rand, et al., 1993). Results indicate that the G-6-PDH is not osmotically sensitive and thus, the change in the number of PEG-inaccessible water molecules (ANw) measured in the coupled reaction is only the difference between the glucose-bound and glucosefree conformations of HK. ANw ~ 450 with PEGs of MW > 2000 under conditions for both binding (Reid and Rand, 1997) and kinetic assays. The contribution water may play in the binding of ATP (Km = 0.24 + 0.02 mM) has also been examined. It was found that in this case ANw = (for osmotic pressures < 2.8x10* dynes/cm^), suggesting no additional numbers of waters are displaced when ATP binds to HK. Osmotic pressure experiments were also performed with dimeric HK. It was determined that both the monomeric and dimeric forms of HK give the same ANw under low pressures. If this large ANw is due to conformational flexibility, it would appear that the flexibility is not reduced upon dimerization ofthe enzyme.

Enhanced pH and thermal stabilities of invertase immobilized on montmorillonite K-10

Invertase was adsorbed onto micro-porous acid-activated montmorillonite clay (K-10) by two procedures, namely adsorption and covalent binding. The immobilized enzymes were characterized by XRD, surface area measurements and 27Al NMR. XRD measurements revealed an expansion of clay layers due to immobilization which suggests that intercalation had taken place. Surface area measurements also support this observation. 27Al NMR showed that interaction of enzyme with tetrahedral and octahedral Al changes with the immobilization procedure. Sucrose hydrolysis was performed in a batch reactor. The immobilized enzymes showed enhanced pH and thermal stabilities. Optimum pH and temperature were found to increase upon immobilization. The effectiveness factor (η) and Michaelis constant (Km) suggest that diffusional resistances play a major role in the reaction. The immobilized invertase could be stored in buffer of pH 5 and 6 at 5 °C without any significant loss in activity for 20 days.