Trp89 in the lid ofHumicola lanuginosa lipase is important for efficient hydrolysis of tributyrin

To determine whether Trp89 located in the lid of the lipase (EC 3.1.1.3) fromHumicola lanuginosa is important for the catalytic property of the enzyme, site-directed mutagenesis at Trp89 was carried out. The kinetic properties of wild type and mutated enzymes were studied with tributyrin as substrate. Lipase variants in which Trp89 was changed to Phe, Leu, Gly or Glu all showed less than 14% of the activity compared to that of the wild type lipase. The Trp89Glu mutant was the least active with only 1% of the activity seen with the wild type enzyme. All Trp mutants had the same binding affinity to the tributyrin substrate interface as did the wild type enzyme. Wild type lipase showed saturation kinetics against tributyrin when activities were measured with mixed emulsions containing different proportions of tributyrin and the nonionic alkyl polyoxyethylene ether surfactant, Triton DF-16. Wild type enzyme showed a V_max=6000±300 mmol·min⁻¹·g⁻¹ and an apparent K_m=16±2% (vol/vol) for tributyrin in Triton DF-16, while the mutants did not show saturation kinetics in an identical assay. The apparent K_m for tributyrin in Triton DF-16 was increased as the result of replacing Trp89 with other residues (Phe, Leu, Gly or Glu). The activities of all mutants were more sensitive to the presence of Triton DF-16 in the tributyrin substrate than was wild type lipase. The activity of the Trp89Glu mutant was decreased to 50% in the presence of 2 vol% Triton DF-16 compared to the activity seen with pure tributyrin as substrate. Wild type lipase and all mutants except Trp89Glu had the same affinity for the substrate interface formed by 15.6 vol% tributyrin in Triton DF-16. The Trp89Glu mutant showed a lower affinity than all the other lipase variants for the interface of 15.6 vol% tributyrin in Triton DF-16. The study showed that Trp89 located in the lid ofH. lanuginosa lipase is important for the efficient hydrolysis of tributyrin and that this residue plays a role in the catalytic steps after adsorption of the lipase to the substrate interface.     

Comparative Biochemistry of Four Polyester (PET) Hydrolases**

The potential of bioprocessing in a circular plastic economy has strongly stimulated research into the enzymatic degradation of different synthetic polymers. Particular interest has been devoted to the commonly used polyester, poly(ethylene terephthalate) (PET), and a number of PET hydrolases have been described. However, a kinetic framework for comparisons of PET hydrolases (or other plastic-degrading enzymes) acting on the insoluble substrate has not been established. Herein, we propose such a framework, which we have tested against kinetic measurements for four PET hydrolases. The analysis provided values of k_cat and K_M, as well as an apparent specificity constant in the conventional units of M⁻¹s⁻¹. These parameters, together with experimental values for the number of enzyme attack sites on the PET surface, enabled comparative analyses. A variant of the PET hydrolase from Ideonella sakaiensis was the most efficient enzyme at ambient conditions; it relied on a high k_cat rather than a low K_M. Moreover, both soluble and insoluble PET fragments were consistently hydrolyzed much faster than intact PET. This suggests that interactions between polymer strands slow down PET degradation, whereas the chemical steps of catalysis and the low accessibility associated with solid substrate were less important for the overall rate. Finally, the investigated enzymes showed a remarkable substrate affinity, and reached half the saturation rate on PET when the concentration of attack sites in the suspension was only about 50 nM. We propose that this is linked to nonspecific adsorption, which promotes the nearness of enzyme and attack sites.     

The kinetics of enzyme catalyzed synthesis of sterol ester

The production of sterol ester by transesterification of β‐sitosterol with fish oil (TAG) catalyzed by Thermomyces lanuginosus immobilized lipase enzyme with varied reaction parameters such as temperature, substrate molar ratio, concentration of enzyme to deduce the enzyme kinetics for the reaction was investigated. For this transesterification reaction, the kinetic model was derived by using Ping Pong Bi Bi Mechanism. The K_m value from the first plot containing fish oil as substrate was 1.31 ± 0.05, while K_m from the second plot containing β‐sitosterol as substrate was 1.01 ± 0.04; identical V_max (0.213 ± 0.06) values were obtained by keeping one of the substrate concentration constant and varying the other. Practical applications : Deduction of reaction kinetics is an important criterion to ascertain the viability of any chemical process. Enzymatic processes need special attention to set model reaction parameters which could help in optimization or design of the actual process. In the present study we have derived the enzyme kinetics for the production sterol ester, an important nutraceutical, and calculated its K_m and V_max values along with the Arhenius activation energy to establish the viability of the reaction.     

Bovine Brain Diacylglycerol Lipase: Substrate Specificity and Activation by Cyclic AMP-dependent Protein Kinase

Diacylglycerol lipase (EC 3.1.1.3) was purified from bovine brain microsomes using multiple column chromatographic techniques. The purified enzyme migrates as a single band on SDS-PAGE and has an apparent molecular weight of 27 kDa. Substrate specificity experiments using mixed molecular species of 1,2-diacyl-sn-glycerols indicate that low concentrations of Ca²⁺ and Mg²⁺ have no direct effect on enzymic activity and 1,2-diacyl-sn-glycerols are the preferred substrate over 1,3-diacyl-sn-glycerols. The enzyme hydrolyzes stearate in preference to palmitate from the sn-1 position of 1,2-diacyl-sn-glycerols. 1-O-Alkyl-2-acyl-sn-glycerols are not a substrate for the purified enzyme. The native enzyme had a V _max value of 616 nmol/min mg protein. Phosphorylation by cAMP-dependent protein kinase resulted in a threefold increase in catalytic throughput (V _max = 1,900 nmol/min mg protein). The substrate specificity and catalytic properties of the bovine brain diacylglycerol lipase suggest that diacylglycerol lipase may regulate protein kinase C activity and 2-arachidonoyl-sn-glycerol levels by rapidly altering the intracellular concentration of diacylglycerols.     

Effect of pressure on the extractive biocatalysis of ethanol

The effect of pressure on the esterification reaction of ethanol with water-immiscible organic acids, catalysed by a lipase from Mucor miehei (pH 4.5; 30°C), was studied through analysis of the kinetics and equilibrium parameters. An increase of the ethanol distribution between the aqueous and organic phases was observed by the addition of lipase and the increase of the pressure in the system. Furthermore, the enzyme showed high specificity for the acid substrate, esterifying preferentially long chain fatty acids (C₈-C₁₈). In the studies described oleic acid was used as substrate for the esterification reaction. A kinetic study with the free enzyme, showed that pressure affected the extraction system, increasing the maximum reaction rate (> V_max), the affinity (< K_m) and the specificity (> V_max/K_m = k_sp) of the enzyme to the substrate, probably due to the effect of pressure on the electrostatic interactions in biological systems. The enzyme operational stability, at 30°C, improved significantly with the increase of pressure, having lower values for the deactivation constant (k) (8.3 × 10^?3 h^?1) and higher values for the half-life times (t_1/2) (77 h) in comparison with those obtained under atmospheric pressure conditions (k = 2.3 × 10^?2h^?1; t_1/2 = 30 h).     

Site-directed mutagenesis of a highly active Staphylococcus epidermidis lipase fragment identifies residues essential for catalysis

A fragment of Staphylococcus epidermidis lipase gene (Lys-303 to Lys-688) was inserted into plasmid pET-20b(+). The resulting C-terminal His-tagged recombinant protein (43 kDa) was overexpressed in Escherichia coli BL21(DE3) as a highly active lipase and was purified with nickel-coupled resin. Putative catalytic sites were determined by site-directed mutagenesis. Mutant enzymes (S418C and H648K) lost enzyme activities, which strongly suggests that the proposed residues of Ser-418 and His-648 are involved in catalysis. Site-directed mutagenesis showed that in comparison with wild-type enzyme, the M419A and V649l enzymes showed a 2.0- and 4.0-fold increase in the k _cat/K _m′ respectively, but the M419l, M419Q, V649A, and V649L variants lost enzyme activities. The wild-type enzyme and the V649l mutant favored the hydrolysis of p-nitrophenyl esters of butyrate, but the M419A favored decanoate. The results suggested that the amino acid residues (Met-419 and Val-649), following the catalytic triad, could affect the substrate specificity and/or catalytic efficiency. This work was presented at the Biocatalysis Symposium in April 2000, held at the 91st Annual Meeting and Expo of the American Oil Chemists' Society, San Diego, CA.     

Hydrolysis of used frying palm olein and sunflower oil catalyzed by porcine pancreatic lipase

The enzymatic hydrolysis of frying used vegetable oils with different degrees of alteration were measured using porcine pancreatic lipase (acylglycerol acylhydrolase EC 3.1.1.3). Successive frying of potatoes significantly increased the level of total polar lipid content in the palm olein from 9.3±0.1 mg/100 mg oil to 26.4±0.3 mg/100 mg oil after 90 fryings, and from 4.0±0.1 mg/100 mg oil to 27.7±0.3 mg/100 mg oil in sunflower oil after 60 fryings. Triacylglycerol polymers, triacylglycerol dimers, and oxidized triacylglycerols also increased 37-, 7.9-, and 7.5-times in palm olein, respectively, and 56-, 22-, and 4.7-times in sunflower oil, respectively. However, diacylglycerols and free fatty acid levels related to hydrolytic alteration did not increase with the number of fryings in both oils. The substrate concentration in the reactor was determined by calculating the molecular weight of each oil showing a different degree of alteration. We compared the methodology used by us and that used by other authors. The results show that the methods are reproducible and that the values obtained are in concordance with theoretical values. The kinetic parameters apparent Michaelis-Menten constant (K _M ^app ) and apparent maximum velocity of hydrolysis (V _max ^app ) were different in unused palm olein (5.1±0.7 and 166±7.6, respectively) than in sunflower oil (2.2±0.3 and 62±2.2, respectively). However, changes inK _M ^app andV _max ^app were not related to the degree of alteration of the oils.     

Kinetic data and substrate specificity of a keratinase from Chryseobacterium sp. strain kr6

BACKGROUND: Keratinases are important enzymes for biotechnological processes involving keratin hydrolysis. In this work substrate specificity and kinetic properties of a keratinase from Chryseobaterium sp. were investigated. RESULTS: The optimal conditions for activity of purified keratinase with respect to pH, temperature and sodium chloride concentration were established using factorial design and surface response techniques. The optimum conditions for keratinase activity were pH from 7.4 to 9.2, temperature from 35 °C to 50 °C and NaCl concentration from 50 to 340 mmol L^?1, having azocasein as substrate. Subsequently, the kinetic parameters for this substrate were determined to be K_m = 0.75 mg mL^?1 and V_max = 59.5 U min^?1. The K_i value for 1,10‐phenanthroline was estimated at 0.78 mmol L^?1. The enzyme specificity was evaluated over different synthetic and insoluble substrates. The protease exhibited specificity with selectivity for hydrophobic and positively charged residues. In relation to the insoluble substrates, the enzyme hydrolyzed preferably chicken nails. CONCLUSIONS: This enzyme effectively hydrolyzes insoluble keratin substrates. The knowledge of keratinase properties is an essential step in the development of biotechnological processes involving keratin hydrolysis. Copyright © 2008 Society of Chemical Industry     

Purification and characterization of an extracellular lipase from <Emphasis Type="Italic">Geotrichum marinum</Emphasis>

An extracellular lipase (EC 3.1.1.3) from Geotrichum marimum was purified 76-fold with 46% recovery using Octyl Sepharose 4 Fast Flow and Bio-Gel A 1.5 m chromatography. The purified enzyme showed a prominent band on SDS-PAGE and a single band on native PAGE based on the activity staining. The molecular mass of the lipase was estimated to be 62 kDa using SDSPAGE and Bio-Gel A chromatography, indicating that the lipase likely functions as a monomer. The pl of the lipase was determined to be 4.54. The apparent V _max and K _m were 1000 μmol/min/mg protein and 11.5 mM, respectively, using olive oil emulsified with taurocholic acid as substrate. The lipase demonstrated a pH optimum at pH 8.0 and a temperature optimum at 40°C. At 6 mM, Na⁺, K⁺, Ca²⁺, and Mg²⁺ stimulated activity, but Na⁺, and K⁺ at 500 mM and Fe²⁺ and Mn²⁺ at 6 mM reduced lipase activity. The anionic surfactant, taurocholic acid, and the zwitterionic surfactant, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, enhanced the activity at 0.1 mM. Other anionic surfactants such as SDS and sodium dioctyl sulfosuccinate, the cationic surfactants methylbenzethonium bromide and cetyltriethylammonium bromide, and the nonionic surfactants Tween-20 and Triton X-100 inhibited the lipase activity to different extents. The lipase was found to have a preference for TG containing cis double bonds in their FA side chains, and the reaction rate increased with an increasing number of double bonds in the side chain. The lipase had a preference for ester bonds at the sn-1 and sn-3 positions over the ester bond at the sn-2 position.     

Application of chitosan‐entrapped β‐galactosidase in a packed‐bed reactor system

The model enzyme β‐galactosidase was entrapped in chitosan gel beads and tested for hydrolytic activity and its potential for application in a packed‐bed reactor. The chitosan beads had an enzyme entrapment efficiency of 59% and retained 56% of the enzyme activity of the free enzyme. The Michaelis constant (K_m) was 0.0086 and 0.011 μmol/mL for the free and immobilized enzymes, respectively. The maximum velocity of the reaction (V_max) was 285.7 and 55.25 μmol mL^?1 min^?1 for the free and immobilized enzymes, respectively. In pH stability tests, the immobilized enzyme exhibited a greater range of pH stability and shifted to include a more acidic pH optimum, compared to that of the free enzyme. A 2.54 × 16.51‐cm tubular reactor was constructed to hold 300 mL of chitosan‐immobilized enzyme. A full‐factorial test design was implemented to test the effect of substrate flow (20 and 100 mL/min), concentration (0.0015 and 0.003M), and repeated use of the test bed on efficiency of the system. Parameters were analyzed using repeated‐measures analysis of variance. Flow (p < 0.05) and concentration (p < 0.05) significantly affected substrate conversion, as did the interaction progressing from Run 1 to Run 2 on a bed (p < 0.05). Reactor stability tests indicated that the packed‐bed reactor continued to convert substrate for more than 12 h with a minimal reduction in conversion efficiency. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 91: 1294–1299, 2004     

Immobilization of pepsin on an acrylamide/2‐hydroxyethyl methacrylate copolymer and its use in casein hydrolysis

Pepsin was immobilized through covalent bonding on a copolymer of acrylamide and 2‐hydroxyethyl methacrylate via the individual and simultaneous activation of both groups. The extent of enzyme coupling upon the activation of both the amino and hydroxyl groups of the copolymer resulted in a synergistic effect. However, the order of activation of the support was critical. The covalently bound enzyme retained more than 50% of its activity even after six cycles. The storage stability of the covalently bound enzyme was 60% after storage for 1 month, whereas the free enzyme lost all of its activity within 10 days of storage at 35°C. The Michaelis constant (K_m) and maximum reaction velocity (V_max) were 1.1 × 10^?6 and 0.87 for the free enzyme and 1.2 × 10^?6 and 0.98 for the covalently bound enzyme when the enzyme concentration was kept constant and the substrate concentration was varied. Similarly, K_m and V_max were 6.73 × 10^?11 and 0.47 for the free enzyme and 7.59 × 10^?11 and 0.545 for the covalently bound enzyme when the substrate concentration was kept constant and the enzyme concentration was varied; this indicated no conformational change during coupling, but the reaction was concentration‐dependent. The hydrolysis of casein was carried out with a fixed‐bed reactor (17 cm × 1 cm). Maximum hydrolysis (90%) was obtained at a 2 cm³/min flow rate at 35°C with a 1 mM casein solution. © 2005 Wiley Periodicals, Inc. J Appl Polym Sci 96: 1544–1549, 2005     

Fourier-transform infrared assay of bile salt-stimulated lipase activity in reversed micelles

A Fourier-transform infrared (FT-IR) spectroscopic method has been developed for assaying the bile salt-stimulated human milk lipase (BSSL, EC3.1) catalyzed hydrolysis of triolein in AOT reversed micelles in iso-octane. At 37°C in 50 mmol dm^?3 AOT the molar absorbtivities for the carbonyl stretching frequencies for triolein (at 1751 cm^?1) and oleic acid (at 1714 cm^?1) were 1646 dm³ mol^?1 cm^?1 and 743 dm^?3 mol^?1 cm^?1, respectively. The rate was linearly dependent upon the concentration of enzyme in the water pool up to 10 mg cm^?3 and maximum activity was observed at a ratio (w₀) of [H₂O]:[AOT] = 16·7. Using these conditions, and in the presence of 10 mmol dm^?3 sodium taurocholate (TC), the derived Michaelis–Menten parameters V_max and K_m were 57·5 μmol min^?1 mg^?1 and 5·53 mmol dm^?3, respectively. These results are compared with those obtained in an oil-in-water microemulsion system and are discussed in terms of the relative partitioning of the enzyme and the substrate in the aqueous and oil phases and the interfacial concentration of the substrate in the two systems.     

Enzymatic Synthesis of Thioesters in Non-conventional Solvents

The feasibility of enzymatic thioesterification between oleic acid and butanethiol in n-hexane, with the immobilised lipase (Lipozyme) from Mucor miehei, has been demonstrated. The immobilised enzyme quantity (100 mg), temperature (40°C), pH range (6–9) and water content (10%) were studied and their optimum values were determined. A preliminary kinetic study indicated a low butanethiol affinity for the enzyme (K_m = 1·85 mol dm⁻³). Even when butanethiol was used without solvent, no substrate inhibition was observed. The possibility of carrying out this reaction in a natural solvent, supercritical carbon dioxide (SCCO₂), was successfully verified. After 8 h reaction, a conversion yield of oleic acid of about 33% was obtained. © 1997 SCI.     

Characterization of Candida rugosa Lipase Immobilized on Poly(N‐methylolacrylamide) and Its Application in Butyl Butyrate Synthesis

Candida rugosa lipase was immobilized on poly(N‐methylolacrylamide) by physical adsorption. The biocatalyst performance (immobilized lipase) was evaluated in both aqueous (hydrolysis) and organic (butyl butyrate synthesis) media. In the first case, a comparative study between free and immobilized derivatives was provided in terms of pH, temperature and thermal stability following the olive oil hydrolysis, establishing new optimum values. In the second case, the influence of temperature, biocatalyst concentration and acid/alcohol molar ratio was simultaneously studied according to a 2³ full experimental design. The highest molar conversion (96 %), volumetric productivity (1.73 g L^–1 h^–1) and specific esterification activity (1.00 μM mg^–1 min^–1) were obtained when working at the lowest level of temperature and butyric acid in excess. Under these conditions, repeated batch use of the immobilized enzyme was performed and half‐life time (t_1/2) was found to be 145 h.     

Maximum volumetric production of β‐galactosidase by Kluyveromyces fragilis in fed‐batch culture with automatic control

The production of β‐galactosidase by Kluyveromyces fragilis was studied in different culture systems, with dissolved oxygen concentration control and using defined media. An operating strategy of fed‐batch culture with automatic control of substrate addition regulated by dissolved oxygen concentration, consisting of the replacement of variable volumes of broth by fresh medium (once the fed‐batch culture has finished), was designed. The volumetric enzyme productivity (Q_p, 13 600 UI dm^?3 h^?1) obtained was 38% higher than that reached in continuous culture of K fragilis with dissolved oxygen concentration control and far higher than that obtained by batch culture of K fragilis under the same aeration conditions. © 2002 Society of Chemical Industry     

Enzymatic acyl modification of phosphatidylcholine using immobilized lipase and phospholipase A2

Ethanol‐soluble (ES) lecithin mainly contains phosphatidylcholine (PC). The incorporation of caprylic acid into PC using immobilized phospholipase A₂ (PLA₂) and lipase was investigated. The Rhizomucor meihei lipase and the porcine pancreatic PLA₂ were immobilized on the hydrophobic resin Diaion HP‐20 and the modification was carried out in hexane as solvent. HPTLC with densitometer technique was successfully used for monitoring the production of structured phospholipids (PL) (ML‐type PC, MM‐type PC, and lysophosphatidylcholine; L: long‐chain fatty acid, M: medium‐chain fatty acid). The various parameters such as the effects of reaction temperature, enzyme loading, and the effect of molar proportion of substrate were studied in order to determine the optimum reaction conditions for the acidolysis reaction. The optimal operating conditions for the PLA₂‐catalyzed reaction were obtained as 50°C temperature, 50% (wt/wt of substrate) enzyme loading, and a 1:12 molar proportion of PC/caprylic acid. For the lipase‐catalyzed reaction, the optimized temperature was the same as for PLA₂, but the enzyme loading and molar proportion were slightly lower, i.e., 40 % w/w of substrate and 1:9 PC/caprylic acid, respectively. The effects of these parameters on the production of structured PL were compared. Under these optimal conditions, the ML‐type PC content was higher in the PLA₂‐catalyzed reaction, i.e., 45.29 mol%, and in the lipase‐catalyzed reaction it was 38.74 mol%.     

The essential oil fromTagetes minuta L. modulates the binding of [3H]flunitrazepam to crude membranes from chick brain

The hypothesis that the essential oil fromTagetes minuta L. can interact with biological membranes was investigated by assessing its ability of perturbing the binding of a benzodiazepine [flunitrazepam (FNTZ)] to crude membranes from chick brains. The essential oil fromT. minuta L. inhibited [³H]FNTZ specific binding to chick brain membranes. These values were obtained from the analysis of the saturation curve for the kinetic parameters: dissociation equilibrium constant (K_d)=2.47±0.32 nM, maximal binding (B_max)=556±5 fmoles/mg protein, and Hill coefficient (n)=1.00±0.07 in the absence, and K_d=6.73±1.4 nM, B_max=583±69 fmoles/mg protein, and n=1.02±0.08 in the presence of 29 μg/mL of essential oil. The essential oil could self-aggregate with a critical micellar concentration (CMC) of 60 μg/mL. The marked increase in [³H]FNTZ nonspecific binding starting at 60 μg of essence per mL was due to that phenomenon and revealed the ability of self-aggregated structures to interact with membranes. [³H]FNTZ specific binding decrement as a function of essence concentration cannot be ascribed merely to oil's micelles ability of trapping the lipophilic radioligand molecules, because the discontinuous behavior that characterizes a monomer-aggregate phase transition was not shown. Oil's components might behave as competitive inhibitors or allosteric modulators of FNTZ specific binding. However, their ability to increase FNTZ nonspecific binding at concentrations below oil's CMC suggests that this effect may be due to oil's partitioning into the lipid bilayer. This latter phenomenon would induce an increment in membrane fluidity and a change on FNTZ binding site toward a lower affinity conformation. Therefore, the essential oil components can interact with brain membranes either as monomers, by partitioning into the lipid bilayer, or as self-aggregated structures, through an adsorption to the membrane surface.     

Production of lipase from Penicillium sp. using waste oils and Nopalea cochenillifera

The present communication deals with the production of lipase from Penicillium sp. using waste oils and palm cactus (Nopalea cochenillifera) especially as nutrient source of low cost. Two different waste oils were tested: waste frying oil from an industrial kitchen and waste lubricating oil (WLO) from a gas station. Using Doehlert experimental design and response surface methodology, the optimum conditions for lipase production were 96?h fermentation, WLO as the inductor, with specific activity of 0.22?UA?mg^?1. The enzyme was able to remain with more than 58% of its original activity until 30?min at 60°C. The kinetic constants were K_m?=?9.93?mM and V_max?=?2.58 UA min^?1 using p-nitrophenyl palmitate (p-NPP) as substrate. Results showed that Penicillium sp. was able to produce lipase from waste oils using N. cochenillifera, thus having biotechnological potential in waste oil biotransformation.     

Biocatalytic Properties of Lipase from Walnut Seed (<Emphasis Type="Italic">Juglans regia</Emphasis> L.)

Lipase (E.C. 3.1.1.3) from walnut seed was purified 28.6-fold with 31% yield using Sephadex G-100 gel chromatography. Olive oil served as good substrate for the enzyme. The optimum pH and temperature were 9.0 and 70 °C, respectively. The lipase was stable between 30 and 80 °C for 5 min. K _m and V _max values were determined as 48 mM and 23.06 × 10⁻³ U/min mg for triolein as substrate. Lipase activity was slightly reduced by Cu²⁺, Ca²⁺, Hg²⁺, Mn²⁺, and Ni²⁺ ions, while Mg²⁺ and Zn²⁺ had no effects. Anionic surfactant sodium dodecyl sulfate stimulated lipase activity while non-ionic surfactants Tween-80 and Triton X-100 had negligible effects on enzymatic activity. The enzyme activity was not affected by 50 mM urea and thioacetamide. Potassium ferricyanide, n-bromosuccinamide and potassium cyanide reduced the enzyme activity. The enzyme showed a good stability in organic solvents, the best result being in n-hexane (113% residual activity). The activity of dialysate was maintained approximately 80% for 1 year at −20 °C.     

Kinetic Analysis of PRMT1 Reveals Multifactorial Processivity and a Sequential Ordered Mechanism

Arginine methylation is a prevalent post‐translational modification in eukaryotic cells. Two significant debates exist within the field: do these enzymes dimethylate their substrates in a processive or distributive manner, and do these enzymes operate using a random or sequential method of bisubstrate binding? We revealed that human protein arginine N‐methyltransferase 1 (PRMT1) enzyme kinetics are dependent on substrate sequence. Further, peptides containing an Nη‐hydroxyarginine generally demonstrated substrate inhibition and had improved K_M values, which evoked a possible role in inhibitor design. We also revealed that the perceived degree of enzyme processivity is a function of both cofactor and enzyme concentration, suggesting that previous conclusions about PRMT sequential methyl transfer mechanisms require reassessment. Finally, we demonstrated a sequential ordered Bi–Bi kinetic mechanism for PRMT1, based on steady‐state kinetic analysis. Together, our data indicate a PRMT1 mechanism of action and processivity that might also extend to other functionally and structurally conserved PRMTs.