Steady-State Kinetics of Aminopeptidase Catalysis: A Stopped-Flow Radiationless Energy Transfer Study

Stopped-flow radiationless energy transfer experiments have been carried out to investigate the hydrolysis of some dansyl peptide substrates (S) catalyzed by aminopeptidase (E). RET between enzyme tryptophanyl residues and the dansyl group in the substrate allowed direct observation and quantitation of the enzyme-substrate (ES) complexes. Analysis of the stopped-flow RET traces gives k_cat = 1.32 s^?1 and K_M = 47 μM for Leu-Ala-NH(CH₂)₂NH-Dns (Leu-Ala-DED) and k_cat = 4.80 s^?1 and K_M = 196 μM for Leu-Gly-NH(CH₂)₂NH-Dns (Leu-Gly-DED). The activation energies of the enzymatic reactions were determined from the Arrhenius plots to be 57 and 38 kJ mol^?1 for Leu-Ala-DED and Leu-Gly-DED, respectively. The kinetic results indicate that the enzyme binds Leu-Ala-DED more tightly than Leu-Gly-DED as revealed by a small value of K_M. That this enzyme catalyzes the turnover of Leu-Gly-DED more efficiently than Leu-Ala-DED is reflected in a large value of k_cat and a small activation energy. The RET signals during the hydrolysis of Leu-Val-NH(CH₂)₂NH-Dns were extremely weak probably because of the inefficient energy transfer in the ES complex or the retention of the product in the enzyme after completion of the reaction. Aminopeptidase was inactive towards the dansyl compounds of the single amino acid studied. This fact may be due to an unfavorable conformation of these compounds in the ES complexes (small k_cat) or a weak binding of the substrates to the enzyme (large K_M) or both.     

Catalytic self-assembled monolayers on Au nanoparticles: the source of catalysis of a transphosphorylation reaction

The catalytic activity of a series of Au monolayer protected colloids (Au MPCs) containing different ratios of the catalytic unit triazacyclononane?Zn^II (TACN?Zn^II) and an inert triethyleneglycol (TEG) unit was measured. The catalytic self‐assembled monolayers (SAMs) are highly efficient in the transphosphorylation of 2‐hydroxy propyl 4‐nitrophenyl phosphate (HPNPP), an RNA model substrate, exhibiting maximum values for the Michaelis–Menten parameters k_cat and K_M of 6.7×10^?3 s^?1 and 3.1×10^?4 M , respectively, normalized per catalytic unit. Despite the structural simplicity of the catalytic units, this renders these nanoparticles among the most active catalysts known for this substrate. Both k_cat and K_M parameters were determined as a function of the mole fraction of catalytic unit (x ₁ ) in the SAM. Within this nanoparticle (NP) series, k_cat increases up till x ₁ ≈0.4, after which it remains constant and K_M decreases exponentially over the range studied. A theoretical analysis demonstrated that these trends are an intrinsic property of catalytic SAMs, in which catalysis originates from the cooperative effect between two neighboring catalytic units. The multivalency of the system causes an increase of the number of potential dimeric catalytic sites composed of two catalytic units as a function of the x ₁ , which causes an apparent increase in binding affinity (decrease in K_M). Simultaneously, the k_cat value is determined by the number of substrate molecules bound at saturation. For values of x ₁ > 0.4, isolated catalytic units are no longer present and all catalytic units are involved in catalysis at saturation. Importantly, the observed trends are indicative of a random distribution of the thiols in the SAM. As indicated by the theoretical analysis, and confirmed by a control experiment, in case of clustering both k_cat and K_M values remain constant over the entire range of x ₁ .     

Catalytic properties of recombinant class a TEM-1 β-lactamase and its inhibition by sulbactam,tazobactam, and clavulanic acid

A homogeneous preparation of recombinant class A TEM-1 β-lactamase was used in various expression constructs as a genetic marker to provide cell resistance to ampicillin; its kinetic parameters (K _M = 22 μM, V = 0.39 μM/s, k _cat = 31.2 s⁻¹, k _cat/K _M =1/4 μM/s⁻¹) were determined using the chromogenic substrate CENTA. Comparative analysis of the obtained K _M value and the literature data demonstrated that the recombinant enzyme is 3 times more specific against the CENTA substrate than the native enzyme (K _M = 70 μM). Competitive inhibition of recombinant β-lactamase by sulbactam, tazobactam, and clavulanic acid was demonstrated. The CENTA inhibition constants for sulbactam, tazobactam, and clavulanic acid (K _{I (sulbactam)} = 0.43 μM, K _{I (tazobactam)} = 0.041 μM, and K _{I (clavulanic acid)} = 0.046 μM) were determined for the first time. It was shown that tazobactam and clavulanic acids are the most efficient inhibitors of recombinant β-lactamase and produced the same inhibitory effect.     

A Laccase of <Emphasis Type="Italic">Fomes durissimus</Emphasis> MTCC-1173 and Its Role in the Conversion of Methylbenzene to Benzaldehyde

A laccase has been purified from the liquid culture growth medium containing bagasse particles of Fomes durissimus. The method involved concentration of the culture filtrate by ultrafiltration and anion exchange chromatography on diethyl aminoethyl cellulose. The sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) and native polyacrylamide gel electrophoresis both gave single protein band indicating that the enzyme preparation was pure. The molecular mass of the purified laccase determined from SDS-PAGE analysis was 75 kDa. Using 2,6-dimethoxyphenol as the substrate, the determined K _m and k _cat values of the laccase are 182 μM and 0.35 s⁻¹, respectively, giving a k _cat/K _m value of 1.92 × 10³ M⁻¹ s⁻¹. The pH and temperature optimum were 4.0 and 35 °C, respectively. The purified laccase has yellow colour and does not show absorption band around 610 nm found in blue laccases. Moreover, it transformed methylbenzene to benzaldehyde in the absence of mediator molecules, property exhibited by yellow laccases.     

Structure-function relationships of a catalytically efficient β-<Emphasis Type="SmallCaps">D</Emphasis>-xylosidase

β-d-Xylosidase from Selenomonas ruminantium is revealed as the best catalyst known (k _cat, k _cat/K _m) for promoting hydrolysis of 1,4-β-d-xylooligosaccharides. ¹H nuclear magnetic resonance experiments indicate the family 43 glycoside hydrolase acts through an inversion mechanism on substrates 4-nitrophenyl-β-d-xylopyranoside (4NPX) and 1,4-β-d-xylobiose (X2). Progress curves of 4-nitrophenyl-β-d-xylobioside, xylotetraose and xylohexaose reactions indicate that one residue from the nonreducing end of substrate is cleaved per catalytic cycle without processivity. Values of k _cat and k _cat/K _m decrease for xylooligosaccharides longer than X2, illustrating the importance to catalysis of subsites −1 and +1 and the lack there of subsite +2. Homology models of the enzyme active site with docked substrates show that subsites bey ond−1 are blocked by protein and subsites bey ond +1 are not formed; they suggest that D14 and E186 serve catalysis as general base and general acid, respectively. Individual mutations, D14A and E186A, erode k _cat and k _cat/K _m by <10³ and to asimilar extent for substrates 4NPX and 4-nitrophenyl-α-l-arabinofuranoside (4NPA), indicating that the two substrates share the same active site. With 4NPX and 4NPA, pH governs k _cat/K _m with pK _a values of 5.0 and 7.0 assigned to D14 and E186, respectively. k _cat (4NPX) has a pK _a value of 7.0 and k _cat (4NPA) is pH independent above pH 4.0, suggesting that the catalytically inactive, “dianionic” enzyme form (D14-E187-) binds 4NPX but not 4NPA. The mention of firm names or trade products does not imply that they are end orsed or recommended by the US Department of Agriculture over other firms or similar products not mentioned.     

Simulaaneous ethanol and cellobiose inhibition of cellulose hydrolysis studied with integrated equations assuming constant or variable substrate concentration

The integrated forms of the Michaelis-Menten equation assuming variable substrate (depletion) or constant substrate concentration were used to study the effect of the simultaneous presence of two exoglucanase Cel7A inhibitors (cellobiose and ethanol) on the kinetics of cellulose hydrolysis. The kinetic parameters obtained, assuming constant substrate (K _m =21 mM, K _ic =0.035 mM; K _icl =1.5×10¹⁵mM; k _cat=12 h⁻¹) or assuming variable substrate (K _m =16 mM, K _ic =0.037 mM; K _icl =5.8×10¹⁴ mM; k _cat=9 h⁻¹), showed a good similarity between these two alternative methodologies and pointed out that bothethanol and cellobiose are competitive inhibitors. Nevertheless, ethanol is a very weak inhibitor, as shown by the large value estimated for the kinetic constant K _icl . In addition, assuming different concentrations of initial accessible substrate present in the reaction, both inhibition and velocity constants are at the same order of magnitude, which is consistent with the obtained values. The possibility of using this kind of methodology to determine kinetic constants in general kinetic studies is discussed, and several integrated equations of different Michaelis-Menten kinetic models are presented. Also examined is the possibility of determining inhibition constants without knowledge of the true accessible substrate concentration.     

Erratum to: Catalytic Properties of Two Rhizopus oryzae 99-880 Glucoamylase Enzymes Without Starch Binding Domains Expressed in Pichia pastoris

Catalytic properties of two glucoamylases, AmyC and AmyD, without starch binding domains from Rhizopus oryzae strain 99-880 are determined using heterologously expressed enzyme purified to homogeneity. AmyC and AmyD demonstrate pH optima of 5.5 and 6.0, respectively, nearly one unit higher than the Rhizopus AmyA glucoamylase enzyme. Optimal initial activities are at 60 and 50 °C for AmyC and AmyD, respectively. Inactivation of both enzymes occurs at 50 °C following 30 min pre-incubation. The two enzymes demonstrate substantially slower catalytic rates toward soluble starch relative to AmyA. AmyC has similar k _cat and K _m for oligosaccharides to other Rhizopus and Aspergillus glucoamylases; however, the enzyme has a 2-fold lower K _m^maltose. AmyD has a 3-fold higher K _m and lower k _cat for maltooligosaccharides than AmyC and other glucoamylases. AmyC (but not AmyD) exhibits substrate inhibition. K _i for substrate inhibition decreases with increasing length of the oligosaccharides. Data from pre-steady-state binding of AmyC to maltose and maltotriose and pre-steady-state to steady-state catalytic turnover experiments of AmyC acting on maltotriose were used to interrogate models of substrate inhibition. In the preferred model, AmyC accumulates an enzyme-maltose-maltotriose dead-end complex in the steady state.     

Discovery of Selective Small‐Molecule Activators of a Bacterial Glycoside Hydrolase

Fragment‐based approaches are used routinely to discover enzyme inhibitors as cellular tools and potential therapeutic agents. There have been few reports, however, of the discovery of small‐molecule enzyme activators. Herein, we describe the discovery and characterization of small‐molecule activators of a glycoside hydrolase (a bacterial O‐GlcNAc hydrolase). A ligand‐observed NMR screen of a library of commercially available fragments identified an enzyme activator which yielded an approximate 90 % increase in k_cat/K_M values (k_cat=catalytic rate constant; K_M=Michaelis constant). This compound binds to the enzyme in close proximity to the catalytic center. Evolution of the initial hits led to improved compounds that behave as nonessential activators effecting both K_M and V_max values (V_max=maximum rate of reaction). The compounds appear to stabilize an active “closed” form of the enzyme. Such activators could offer an orthogonal alternative to enzyme inhibitors for perturbation of enzyme activity in vivo, and could also be used for glycoside hydrolase activation in many industrial processes.     

A novel approach to measure all rate constants in the simplest enzyme kinetics model

Enzymes play vital roles in life processes. Almost all biochemical reactions are mediated by enzymes. The rate constants of enzyme kinetics are the most important parameters for the reactions catalyzed by enzymes. In 1902, Adrian Brown proposed a simple single-substrate-single-product model which contains only three rate constants k ₁, k ₋₁ and k ₂. So far, biologists can measure the Michaelis constant K _M and the catalytic constant k _cat , which actually is equal to k ₂, according to Michaelis–Menten equation. Using temperature jump method or transient state kinetics, k ₁, k ₋₁ and k ₂ can be determined. However, these methods are complicated. In this article, we design a novel simple method that could determine the rate constants k ₁ and k ₋₁ based on knowing k _cat and K _M . Our numerical experiments show that the three rate constants can be calculated rather precisely. Hence, we believe that biochemists could design experiments to measure the rate constants based on our method. This work was partially supported by the National Natural Science Foundation of China (NSFC) under Grant No. 10771206 and partially by 973 project (2004CB318000) of P. R. China.     

Characterization of a Thermostable Family 1 Glycosyl Hydrolase Enzyme from <Emphasis Type="Italic">Putranjiva roxburghii</Emphasis> Seeds

A 66-kDa thermostable family 1 Glycosyl Hydrolase (GH1) enzyme with β-glucosidase and β-galactosidase activities was purified to homogeneity from the seeds of Putranjiva roxburghii belonging to Euphorbiaceae family. N-terminal and partial internal amino acid sequences showed significant resemblance to plant GH1 enzymes. Kinetic studies showed that enzyme hydrolyzed p-nitrophenyl β-d-glucopyranoside (pNP-Glc) with higher efficiency (K _cat/K _m = 2.27 × 10⁴ M⁻¹ s⁻¹) as compared to p-nitrophenyl β-d-galactopyranoside (pNP-Gal; K _cat/K _m = 1.15 × 10⁴ M⁻¹ s⁻¹). The optimum pH for β-galactosidase activity was 4.8 and 4.4 in citrate phosphate and acetate buffers respectively, while for β-glucosidase it was 4.6 in both buffers. The activation energy was found to be 10.6 kcal/mol in the temperature range 30–65 °C. The enzyme showed maximum activity at 65 °C with half life of ~40 min and first-order rate constant of 0.0172 min⁻¹. Far-UV CD spectra of enzyme exhibited α, β pattern at room temperature at pH 8.0. This thermostable enzyme with dual specificity and higher catalytic efficiency can be utilized for different commercial applications.     

Catalysis of benzisoxazole isomerization by molecularly imprinted polymers

A tailor-made catalytically active polymer catalyzing the benzisoxazole isomerization is described. Kinetic studies carried out in water/ethanol (3:1, v/v) at room temperature, showed a rate acceleration (k_MIP/k_control) of 7.2-fold compared to the control polymer. The imprinted polymer exhibits Michaelis-Menten kinetics with a K_m of 0.484 mM and a k_cat of 0.205 min⁻¹. Compared with the uncatalyzed reaction, a rate enhancement ((k_cat/K_m)/k_uncat) of 4 × 10⁴ fold was obtained. Substrate selectivity, accessible binding site analysis, dissociation constant determination, and inhibition study were also performed.     

Purification of chloroperoxidase from Musa paradisiaca stem juice

Chloroperoxidase from Musa paradisiaca stem juice has been purified to homogeneity using a concentration obtained by ultrafiltration and anion exchange chromatography on diethylaminoethyl (DEAE) cellulose. The purified enzyme gave a single protein band in SDS‐PAGE analysis corresponding to molecular mass of 43 kDa. The native PAGE analysis result has also given a single protein band, confirming the purity of the enzyme. The purified enzyme was chlorinated and brominated with monochlorodimedone, the substrate used for measuring the halogenating activity of chloroperoxidases. The K_m and k_cat values using monochlorodimedone as the substrate were 20 μM and 1.64 s^?1, respectively, giving a k_cat/K_m value of 8.2 × 10⁴ M^?1 s^?1. The pH and temperature optima of the chlorinating activity were 3.0 and 25°C, respectively. The K_m values for the peroxidase activity using pyragallol and H₂O₂ as the variable substrates were 89 and 120 μM, respectively. The pH and temperature optima of the peroxidase activity using pyrogalllol as the substrate were the same as the pH and temperature optima of the halogenating activity. The peroxidase activity of the enzyme is competitively inhibited by sodium azide, indicating that it is a hemeperoxidase different from nonheme peroxidases. © 2012 Wiley Periodicals, Inc. Int J Chem Kinet 45: 92–100, 2013     

Studies towards a tailor-made catalyst for the Diels-Alder reaction using the technique of molecular imprinting

A tailor-made catalytically active polymer catalyzing the bimolecular Diels-Alder reaction is described. Kinetic studies carried out in acetonitrile at 82°C show a 270-fold rate acceleration (k_cat/k_uncat) for the Diels-Alder reaction between tetrachlorothiophene dioxide and maleic anhydride. The imprinted polymer induces Michaelis-Menten kinetics, with an apparent K_m of 42.5 mM and an apparent k_cat of 3.82 × 10⁻² min⁻¹, respectively. Substrate selectivity, accessible binding site analysis, dissociation constant determination, and inhibition study were also performed.     

Novel Activity of UDP-Galactose-4-Epimerase for Free Monosaccharide and Activity Improvement by Active Site-Saturation Mutagenesis

Uridine diphosphogalactose-4-epimerase (UDP-galactose-4-epimerase, GalE, EC 5.1.3.2) mediates the 4-epimerization of nucleic acid-activated galactose into UDP-glucose. To date, no enzyme is known to mediate 4-epimerization of free monosaccharide substrates. To determine the potential activity of GalE for free monosaccharide, Escherichia coli GalE was expressed and purified using Ni-affinity chromatography, and its ability to mediate 4-epimerization of a variety of free keto- and aldohexoses was assessed. Purified GalE was found to possess 4-epimerization activity for free galactose, glucose, fructose, tagatose, psicose, and sorbose at 0.47, 0.31, 2.82, 9.67, 15.44, and 2.08 nmol/mg protein per minute, respectively. No 4-epimerization activity was found for allose, gulose, altrose, idose, mannose, and talose. The kinetic parameters of 4-epimerization reactions were K _m = 26.4 mM and k _cat = 0.0155 min⁻¹ for d-galactose and K _m = 237 mM and k _cat = 0.327 min⁻¹ for d-tagatose. The 4-epimerization of tagatose, a reaction of commercial interest, was enhanced twofold (19.79 nmol/mg protein per minute) when asparagine was exchanged with serine at position 179. The novel activity of GalE for free monosaccharide may be beneficial for the production of rare sugars using cheap natural resources. Potential strategies for developing enhanced GalE with increased 4-epimerization activity are discussed in the context of the above findings and an analysis of a 3D structural model.     

β-d-Xylosidase from Selenomonas ruminantium: Thermodynamics of Enzyme-Catalyzed and Noncatalyzed Reactions

β-d-Xylosidase/α-l-arabinofuranosidase from Selenomonas ruminantium is the most active enzyme known for catalyzing hydrolysis of 1,4-β-d-xylooligosaccharides to d-xylose. Temperature dependence for hydrolysis of 4-nitrophenyl-β-d-xylopyranoside (4NPX), 4-nitrophenyl-α-l-arabinofuranoside (4NPA), and 1,4-β-d-xylobiose (X2) was determined on and off (k _non) the enzyme at pH 5.3, which lies in the pH-independent region for k _cat and k _non. Rate enhancements (k _cat/k _non) for 4NPX, 4NPA, and X2 are 4.3?×?10¹¹, 2.4?×?10⁹, and 3.7?×?10¹², respectively, at 25 °C and increase with decreasing temperature. Relative parameters k _cat ^4NPX/k _cat ^4NPA, k _cat ^4NPX/k _cat ^X2, and (k _cat/K _m)^4NPX/(k _cat/K _m)^X2 increase and (k _cat/K _m)^4NPX/(k _cat/K _m)^4NPA, (1/K _m)^4NPX/(1/K _m)^4NPA, and (1/K _m)^4NPX/(1/K _m)^X2 decrease with increasing temperature.     

Some kinetic features of the hydrolysis of L-asparagine withE. coli asparaginase

The influence of various effectors (methanol, neutral salts) on the kinetic parameters K_M and k_cat has been studied. The hypothesis has been expressed that chloride ions are responsible for the worsening of the binding of the substrate to the enzyme. The temperature dependence of the kinetic parameters K_M and k_cat for the enzymatic hydrolysis of L-asparagine has been obtained. It has been shown that the graph of log k_cat versus 1/T has a break at 30°C. The effective activation energies below and above the critical point are 6.5 and 3.6 kcal/mole, respectively.Institute of Organic Synthesis, Academy of Sciences of the Latvian SSR, Riga. Translated from Khimiya Prirodnykh Soedinenii, No. 5, pp. 707–709, September–October, 1979.     

Some kinetic features of the hydrolysis of L-asparagine withE. coli asparaginase

The influence of various effectors (methanol, neutral salts) on the kinetic parameters K_M and k_cat has been studied. The hypothesis has been expressed that chloride ions are responsible for the worsening of the binding of the substrate to the enzyme. The temperature dependence of the kinetic parameters K_M and k_cat for the enzymatic hydrolysis of L-asparagine has been obtained. It has been shown that the graph of log k_cat versus 1/T has a break at 30°C. The effective activation energies below and above the critical point are 6.5 and 3.6 kcal/mole, respectively.     

Determination of intrinsic properties of immobilized enzymes

Staphylococcal nuclease has been insolubilized, directly through its amino groups, on CNBr-activated Sepharose 2B. For kinetic studies, a small substrate (thymidine 5′-(p-nitrophenyl phosphate) 3′-phosphate) has been used to measure the hydrolytic activity. With this system the absence of diffusional limitation has been proven. Eadie-Hofstee analysis of the data has been employed to determine the intrinsic kinetic constants of the insolubilized enzyme. Thek _cat-pH andK _M−pH profiles and the activation energies are similar for the soluble and for the insolubilized nuclease. At the same time conditions are established in which a stirred batch reactor containing particles of insolubilized nuclease behaves as an open system.     

Cloning, heterologous expression, and characterization of Thielavia terrestris glucoamylase

Thielavia terrestris is a soil-borne thermophilic fungus whose molecular/cellular biology is poorly understood. Only a few genes have been cloned from the Thielavia genus. We detected an extracellular glucoamylase in culture filtrates of T. terrestris and cloned the corresponding glaA gene. The coding region contains five introns. Based on the amino acid sequence, the glucoamylase was 65% identical to Neurospora crassa glucoamylase. Sequence comparisons suggested that the enzyme belongs to the glycosyl hydrolase family 15. The T. terrestris glaA gene was expressed in Aspergillus oryzae under the control of an A. oryzae α-amylase promoter and an Aspergillus niger glucoamylase terminator. The 75-kDa recombinant glucoamylase showed a specific activity of 2.8 μmol/(min·mg) with maltose as substrate. With maltotriose as a substrate, the enzyme had an optimum pH of 4.0 and an optimum temperature of 60°C. The enzyme was stable at 60°C for 30 min. The K _m and k _cat of the enzyme for maltotriose were determined at various pHs and temperatures. At 20°C and pH 4.0, the enzyme had a K _m of 0.33±0.07 mM and a k _cat of (5.5±0.5)×10³ min⁻¹ for maltotriose. The temperature dependence of k _cat /K _m indicated an activation free energy of 2.8 kJ/mol across the range of 20–70°C. Overall, the enzyme derived from the thermophilic fungus exhibited properties comparable with that of its homolog derived from mesophilic fungi.     

Isomeric Effect on the anticancer Behavior of two Zinc (II) complexes based on 3,5‐bis(1‐imidazoly) pyridine: Experimental and Theoretical Approach

Two isomeric Zinc (II) complexes constructed by 3,5‐bis(1‐imidazoly) pyridine has been synthesized and characterized by single crystal X‐ray diffraction, elemental analyses and infrared spectroscopy. The binding mode and ability of complex 1–2 with CT‐DNA were studied by UV and fluorescence spectra. The intrinsic binding constant K_b (K_b1 = 2.305 × 10⁴ M^?1, K_b2 = 3.095 × 10⁴ M^?1) and the observed association constant K_obs (K_obs1 = 1.523*10⁶ M^?1, K_obs2 = 2.057*10⁶ M^?1) indicated that the insertion ability of complex 2 with CT‐DNA is stronger than complex 1. Gel electrophoresis showed that complexes have a good ability to hydrolyze cleavage pBR322 plasmid DNA. The cytotoxicity and apoptosis studies showed that complexes exhibited excellent cytotoxic activity against HeLa cells, especially complex 2 had better growth inhibition than Cisplatin. Molecular docking study simulated the binding model of complexes with DNA (PDB:4av1), showing an imidazole plane of complex 2 can be inserted into a DNA base pair in relative parallel. Both complexes can be used as potential anticancer agents.