Plots Of Initial Velocity Research Articles

A Biochemical Study of α-Amylase Activity in Saliva of Some Libyan Cigarette Smokers

Tobacco consumption alters many biological parameters, including α-amylase activity. This study reports the effect of cigarette smoking on salivary α-amylase activities of some male heavy smokers (100) in apparent good health compared with (40) male non-smokers as control. The salivary α-amylase activity was assayed using the standard colorimetric method. The α-amylase enzyme was characterized from the saliva samples collected from healthy adult smokers and non-smokers (control) to determine the effects of temperature, pH, and substrate concentration on the kinetic parameters of the enzyme. The analysis of the saliva samples showed that there were significant increases (P<0.05) in the α-amylase activity values in the saliva of cigarette smokers when compared with non-smokers at optimum conditions (2.74±0.37 nmol/min/mg-protein and 2.01±0.16 nmol/min/mg-protein, respectively). The Michaelis constant (Km) values of 0.5±0.02 mM and 1.12±0.08 mM observed for non-smokers and smokers were obtained from the double reciprocal plot of initial velocity (1/V) and substrate concentration (1/[S]). The maximum activities obtained at an optimum temperature of 37ºC, and an apparent pH of 7.0 for both subjects were 31.25±8.24 nmol/min/mg protein and 18.10±4.06 nmol/min/mg protein for non-smokers and smokers respectively. Measurements of salivary α-amylase activity could be useful in the early detection and diagnosis of parotitis. Subsequent studies are required to specify the mechanisms responsible for such a risk.

Evaluating Impact Resistance of Aluminum 6061-T651 Plate using Smoothed Particle Hydrodynamics Method

Performing various experimental, theoretical, and numerical investigations for better understanding of behavioural characteristics of metals under impact loading is of primary importance. In this paper, application of smoothed particle hydrodynamics (SPH) method in impact mechanics is discussed and effective parameters on impact strength of an aluminum plate are investigated. To evaluate the accuracy of smoothed particle hydrodynamics method for simulating impact, Recht and Ipson model is first provided thoroughly for both Rosenberg analytical model and smoothed particle hydrodynamics method, and then plots of initial velocity-residual velocity and initial velocity-absorbed energy for target of aluminum 6061-T651 are presented. The derived information and simulation results expresses that the maximum error percentage of smoothed particle hydrodynamics method in compared with Rosenberg analytical model is within an acceptable range. Therefore, the results of smoothed particle hydrodynamics method verify the Rosenberg analytical model with high accuracy. Results reveal that higher initial impact velocity decreases the time of projectile penetration, and so penetration depth and length as well as the local damage rate of plate increases.

15(V/K) kinetic isotope effect and steady-state kinetic analysis for the transglutaminase 2 catalyzed deamidation and transamidation reactions

The Ca2+-dependent deamidation and transamidation activities of transglutaminase 2 (TG2) are important to numerous physiological and pathological processes. Herein, we have examined the steady-state kinetics and 15(V/K) kinetic isotope effects (KIEs) for the TG2-catalyzed deamidation and transamidation of N-Benzyloxycarbonyl-l-Glutaminylglycine (Z-Gln-Gly) using putrescine as the acyl acceptor substrate. Kinetic parameters determined from initial velocity plots are consistent with previously proposed mechanisms. Significant differences in the 15(V/K) KIEs on NH3 release determined for the deamidation (0.2%) and the transamidation (2.3%) of Z-Gln-Gly suggest the rate-limiting steps of TG2 active site acylation are dependent on the presence of the acyl acceptor. We propose a plausible mechanistic explanation where substrate-induced conformational changes may play a role in promoting catalysis.

Extraction, Purification and Characterization of Sprouting Pearl Millet Alpha-Amylase for Biotechnological Applications

The major challenge faced by the biotechnological industries is the suitable source of biocatalysts. This work was aimed at sourcing for α-amylase from pearl millet. Crude α-amylase was extracted from malted pearl millet with 50 mM sodium acetate buffer pH 5.5; 70% ammonium sulphate saturation precipitated protein with highest α-amylase activity. Upon gel filtration, two major peaks were observed and the active fractions were pooled together and characterized. The optimal pH of α-amylase were 4.5 and 5.5 for peak A and B respectively. Both peaks were observed to have the same optimum temperature of 50°C. The Michealis constant (Km) and maximum velocity (Vmax) obtained from the Lineweaver-Burk plot of initial velocity at different substrate concentrations were 0.57 mg/mL and 476.19 μmol/min for peak A and 0.4 mg/mL and 250 μmol/min for peak B. Results from the kinetic parameters (Km and Vmax), optimal pH and temperature of this enzyme suggest that this enzyme could be useful in some biotechnological applications that involves starch hydrolysis such as in starchy food processing, textile industries, brewery industries, etc.

Organophosphorus pesticides stress deranged kinetic values of a few midgut carbohydrases of naiad of Trithemis aurora (burm.) (Odonata: Libellulidae)

The 40 Hrs. treatment of last instar naiad of Trithemis aurora (Burm.) in Chlorphyriphos and Quinalphos pesticides concentrations (LC50 = 5.12 ×10-7 ppm and 7.6 × 10-8 ppm) has shown significant variations in the enzyme kinetic parameters and arrested the enzymatic activity in the midgut tissue of last instar naiad of T. aurora causing deleterious effect on various carbohydrases at standard temp. and pH value. The midgut amylase ( μ and β amylase) showed the change in the velocity of enzymatic reaction under LC50 conc. of chlorpyriphos. The data of initial velocity and substrate concentration were processed to achieve their reciprocal values. These values were plotted and a characteristic Lineweaver Burke straight line was observed from the graph and values of maximum reaction velocity (Vmax) and Michaelis Menten constants (km) were assessed. The present organophosphorus pesticide showed an inhibitory impact on midgut amylase reaction velocity. The double reciprocal plot of initial velocity and substrate concentration after exposing the enzyme under LC50 conc. of chlorphyriphos resulted in varied Vmax and Km. values. These carbohydrase on treatment with LC50 conc. of chlorphyriphos showed an inhibitory change in the reaction velocity. The 1/V and 1/S values were plotted to achieve a characteristic Lineweaver – Burke pattern of Vmax and km values obtained as 5.0 × 10-2 [M] and 2.0 under LC50 chlorphyriphos stress for α amylase. The km and Vmax values were obtained from 0.625 × 10-3 [M] to 1.25 × 10-2 [M] for various other midgut carbohydrases with Vmax value obtained from 0.28 to 5.0 under chlorpyriphos stress. The Quinalphos inhibited the enzymatic efficiencies of various carbohydrases severely and changed Km and Vmax values were found under the pesticidal stress and found as potent uncompetitive inhibitor for enzymes as values compared to the controlled enzymatic reactions by deranging the kinetic values. The Km values determined as on 1/V and 1/S basis found deranged from 1.66 × 10-3 [M] to 10 × 10-2 [M]. The Vmax values were found in a range of 0.41 to 3.3 under LC50 Quinalphos stress for midgut hydrolases. The analysis of enzymec kinetic values revealed the great inhibitory and deranged activities of various carbohydrases under both the pesticide constrain. The present toxicants were found to change the enzymatic velocity negatively. The LC50 concentrations of these toxicants were sufficient to inhibit the activity of present hydrolases as α and β amylase, α glucosidase, α galactosidase, β galactosidase, β frictosidase and α trehalase obtaining a meaningful Lineweaver – Burke line of plotted reciprocals of data of reaction velocity and substrate concentration.

Catalytic site interactions in yeast OMP synthase

The enigmatic kinetics, half-of-the-sites binding, and structural asymmetry of the homodimeric microbial OMP synthases (orotate phosphoribosyltransferase, EC 2.4.2.10) have been proposed to result from an alternating site mechanism in these domain-swapped enzymes [R.W. McClard et al., Biochemistry 45 (2006) 5330–5342]. This behavior was investigated in the yeast enzyme by mutations in the conserved catalytic loop and 5-phosphoribosyl-1-diphosphate (PRPP) binding motif. Although the reaction is mechanistically sequential, the wild-type (WT) enzyme shows parallel lines in double reciprocal initial velocity plots. Replacement of Lys106, the postulated intersubunit communication device, produced intersecting lines in kinetic plots with a 2-fold reduction of kcat. Loop (R105G K109S H111G) and PRPP-binding motif (D131N D132N) mutant proteins, each without detectable enzymatic activity and ablated ability to bind PRPP, complemented to produce a heterodimer with a single fully functional active site showing intersecting initial velocity plots. Equilibrium binding of PRPP and orotidine 5′-monophosphate showed a single class of two binding sites per dimer in WT and K106S enzymes. Evidence here shows that the enzyme does not follow half-of-the-sites cooperativity; that interplay between catalytic sites is not an essential feature of the catalytic mechanism; and that parallel lines in steady-state kinetics probably arise from tight substrate binding.

Uptake kinetics of arsenic by lettuce cultivars under hydroponics

Arsenic (As) uptake ability based on kinetic parameters by two lettuce cultivars; Sijibaiye (SJBY) and Texuanyanlingsun (TXYLS) was investigated in nutrient solution containing eight levels of arsenic (As). Depletion of As from solution was monitored over a period of 24 h at regular time to estimate As uptake kinetics of the plants. Km and Vmax values were evaluated with the aid of the Lineweaver - Burk plot (that is the double reciprocal plot of initial velocity (1/v) versus substrate concentration (1/[s]). Both cultivars were capable to assimilate As(V), although with different kinetic parameters. The values obtained for Km and Vmax varied with plant cultivars. Using Lineweaver- Burk, values of Km and Vmax were found to be much lower in SJBY (26.721 mg L-1 and 0.7737 mgAsg-1 shoot d.wt.hr-1) than that of TXYLS (81.922 mg L-1 and 1.8488 mgAsg-1 shoot d.wt.hr-1), indicating SJBY had the lowest uptake ability than TXYLS plants. Furthermore, TXYLS had the highest Km value than SJBY, confirming that uptake in the plant cell plasma membrane of TXYLS had a higher affinity for As than SJBY plants. The time (24 h) affects the depletion of As in both cultivars. It can be concluded that among the cultivars, SJBY could be finally identified as the most suitable cultivar to be grown in polluted farmland. Therefore, it has been suggested that there is possibility to lower the As concentration in lettuce by selecting and breeding cultivars with less As concentration that can be safely grown in contaminated soils with slight levels of As for safe consumption. Key words: Arsenic (V), cultivars variation, kinetic parameters, Lineweaver Burk, uptake rate.

Determining the affinity and stoichiometry of interactions between unmodified proteins in solution using Biacore

We describe a general Biacore method for measuring equilibrium binding affinities and stoichiometries for interactions between unmodified proteins and their unmodified ligands free in solution. Mixtures of protein and ligand are preequilibrated at different ratios in solution and then analyzed by Biacore using a sensor chip surface that detects only unbound analyte. Performing the Biacore analysis under mass transport limited conditions allows the concentration of unbound analyte to be determined from the initial velocity of binding. Plots of initial velocity versus the concentration of the varied binding partner are fitted to a quadratic binding equation to give the affinity and stoichiometry of binding. We demonstrate the method using soluble Her2 extracellular domain binding to monovalent, bivalent, and trivalent forms of an anti-Her2 antibody. The affinity we measured agrees with that obtained from conventional Biacore kinetic analysis, and the stoichiometries for the resulting 1:1, 1:2, and 1:3 complexes were confirmed by gel filtration with in-line light scattering. The method is applicable over an affinity range of approximately 100pM to 1μM and is particularly useful when there is concern that covalently modifying one or the other binding partner might affect its binding properties or where multivalency might otherwise complicate a quantitative analysis of binding.

Biphasic Kinetic Behavior of E. coli WrbA, an FMN-Dependent NAD(P)H:Quinone Oxidoreductase

The E. coli protein WrbA is an FMN-dependent NAD(P)H:quinone oxidoreductase that has been implicated in oxidative defense. Three subunits of the tetrameric enzyme contribute to each of four identical, cavernous active sites that appear to accommodate NAD(P)H or various quinones, but not simultaneously, suggesting an obligate tetramer with a ping-pong mechanism in which NAD departs before oxidized quinone binds. The present work was undertaken to evaluate these suggestions and to characterize the kinetic behavior of WrbA. Steady-state kinetics results reveal that WrbA conforms to a ping-pong mechanism with respect to the constancy of the apparent Vmax to Km ratio with substrate concentration. However, the competitive/non-competitive patterns of product inhibition, though consistent with the general class of bi-substrate reactions, do not exclude a minor contribution from additional forms of the enzyme. NMR results support the presence of additional enzyme forms. Docking and energy calculations find that electron-transfer-competent binding sites for NADH and benzoquinone present severe steric overlap, consistent with the ping-pong mechanism. Unexpectedly, plots of initial velocity as a function of either NADH or benzoquinone concentration present one or two Michaelis-Menten phases depending on the temperature at which the enzyme is held prior to assay. The effect of temperature is reversible, suggesting an intramolecular conformational process. WrbA shares these and other details of its kinetic behavior with mammalian DT-diaphorase, an FAD-dependent NAD(P)H:quinone oxidoreductase. An extensive literature review reveals several other enzymes with two-plateau kinetic plots, but in no case has a molecular explanation been elucidated. Preliminary sedimentation velocity analysis of WrbA indicates a large shift in size of the multimer with temperature, suggesting that subunit assembly coupled to substrate binding may underlie the two-plateau behavior. An additional aim of this report is to bring under wider attention the apparently widespread phenomenon of two-plateau Michaelis-Menten plots.

Activation and Inhibition of Pyruvate Carboxylase from Rhizobium etli

While crystallographic structures of the R. etli pyruvate carboxylase (PC) holoenzyme revealed the location and probable positioning of the essential activator, Mg(2+), and nonessential activator, acetyl-CoA, an understanding of how they affect catalysis remains unclear. The current steady-state kinetic investigation indicates that both acetyl-CoA and Mg(2+) assist in coupling the MgATP-dependent carboxylation of biotin in the biotin carboxylase (BC) domain with pyruvate carboxylation in the carboxyl transferase (CT) domain. Initial velocity plots of free Mg(2+) vs pyruvate were nonlinear at low concentrations of Mg(2+) and a nearly complete loss of coupling between the BC and CT domain reactions was observed in the absence of acetyl-CoA. Increasing concentrations of free Mg(2+) also resulted in a decrease in the K(a) for acetyl-CoA. Acetyl phosphate was determined to be a suitable phosphoryl donor for the catalytic phosphorylation of MgADP, while phosphonoacetate inhibited both the phosphorylation of MgADP by carbamoyl phosphate (K(i) = 0.026 mM) and pyruvate carboxylation (K(i) = 2.5 mM). In conjunction with crystal structures of T882A R. etli PC mutant cocrystallized with phosphonoacetate and MgADP, computational docking studies suggest that phosphonoacetate could coordinate to one of two Mg(2+) metal centers in the BC domain active site. Based on the pH profiles, inhibition studies, and initial velocity patterns, possible mechanisms for the activation, regulation, and coordination of catalysis between the two spatially distinct active sites in pyruvate carboxylase from R. etli by acetyl-CoA and Mg(2+) are described.

Study on Reaction Kinetics of Lipase-Catalyzed Synthesis of Vanillyl Nonanoate in Acetone Media

The reaction kinetics for synthesis of vanillyl nonanoate (VN) by lipase-catalyzed transesterification of vanillyl alcohol and methyl nonanoate in acetone was investigated in this study. The reaction catalyzed by lipase was carried out as follows: A reaction mixture containing given concentration of substrates (1ml) and lipase Novozyme 435 (20mg) in acetone (1ml) was shaken at 30°C for 10min. The initial velocity of the reaction was calculated according to the concentration of VN detected by high performance liquid chromatography (HPLC), and the kinetic equation was obtained by analysis of the double reciprocal plot of initial velocity versus substrate concentration. The data calculated by the kinetic equation were basically in agreement with the experimental data with a correlation coefficient at 0.997. The inhibitory action of methanol (by-product) in the reaction was also investigated and the results indicated that the concentration of methanol influences both slope and intercept of y-axis of the double reciprocal curve, and the lines intersect on the X-axis. These results revealed that this transesterification follows the Ping-Pong reaction mechanism.

Recombinant expression, purification and characterization of Bombyx mori (Lepidoptera: Bombycidae) pyridoxal kinase

Pyridoxal kinase (PLK; EC 2.7.1.35) is a key enzyme in the metabolism of vitamin B6 (VB6) in Bombyx mori. A fusion expressional vector pET-22b-BPLK-His was constructed using a sub-cloning technique, the recombinant B. mori PLK was then expressed in Escherichia coli, purified and characterized. Bioinformatics were used to deduce the protein structure and genomic organization of this enzyme. Using Ni Sepharose affinity column chromatography, the recombinant protein was purified to very high degree (approximately 90%). The recombinant PLK exhibits a high specific enzymatic activity (1800 nmol/min/mg of protein). The maximum catalytic activity of this enzyme was recorded over a narrow pH range (5.5-6.0) and Zn2+ is the most effective cation for catalysis under saturating substrate concentrations. When only triethanolamine is present as the cation, K+ is an activator of PLK. A double reciprocal plot of initial velocity suggests that the enzyme catalyses the reaction by means of a sequential catalytic mechanism. Under optimal conditions, the Km value for the substrates of ATP and pyridoxal are 57.9 ± 5.1 and 44.1 ± 3.9 µM. B. mori's genome contains a single copy of the PLK gene, which is 7.73 kb long and contains five exons and four introns, and is located on the eighth chromosome. The PLK may be a dimer with two identical subunits under native conditions, and it is hypothesized that each monomer contains eight α-helices (α1-8), nine β-strands (β1-9) and two segments of 310 helices.

Glutamates 78 and 122 in the Active Site of Saccharopine Dehydrogenase Contribute to Reactant Binding and Modulate the Basicity of the Acid-Base Catalysts

Saccharopine dehydrogenase catalyzes the NAD-dependent oxidative deamination of saccharopine to give l-lysine and alpha-ketoglutarate. There are a number of conserved hydrophilic, ionizable residues in the active site, all of which must be important to the overall reaction. In an attempt to determine the contribution to binding and rate enhancement of each of the residues in the active site, mutations at each residue are being made, and double mutants are being made to estimate the interrelationship between residues. Here, we report the effects of mutations of active site glutamate residues, Glu(78) and Glu(122), on reactant binding and catalysis. Site-directed mutagenesis was used to generate E78Q, E122Q, E78Q/E122Q, E78A, E122A, and E78A/E122A mutant enzymes. Mutation of these residues increases the positive charge of the active site and is expected to affect the pK(a) values of the catalytic groups. Each mutant enzyme was completely characterized with respect to its kinetic and chemical mechanism. The kinetic mechanism remains the same as that of wild type enzymes for all of the mutant enzymes, with the exception of E78A, which exhibits binding of alpha-ketoglutarate to E and E.NADH. Large changes in V/K(Lys), but not V, suggest that Glu(78) and Glu(122) contribute binding energy for lysine. Shifts of more than a pH unit to higher and lower pH of the pK(a) values observed in the V/K(Lys) pH-rate profile of the mutant enzymes suggests that the presence of Glu(78) and Glu(122) modulates the basicity of the catalytic groups.

Mercaptopyruvate Inhibits Tissue-Nonspecific Alkaline Phosphatase and Calcium Pyrophosphate Dihydrate Crystal Dissolution

The enzymatic activities of tissue-nonspecific alkaline phosphatase (TNAP) including capacity to inhibit calcium pyrophosphate dihydrate (CPPD) crystal dissolution are known to be inhibited by endogenous amino acids, notably cysteine. As cysteine is recognized as a strong TNAP inhibitor, we investigated whether cysteine-related metabolites such as mercaptopyruvate (MPA) could show similar enzyme inhibition effects and, if so, whether these effects might be synergistic with cysteine at approximate physiologic concentrations of the amino acids. We studied the inhibitory effects of MPA as well as MPA and cysteine combined in equimolar concentrations on TNAP's phosphatase, inorganic pyrophosphatase, and CPPD crystal dissolution activities. Kinetic parameters V(max), K(M), concentration for 50% inhibition (I(50)), inhibitor constant (K(I)), and specific activities calculated from initial velocity, Eadie-Hofstee, Simple, Dixon, and secondary plots were used to assess enzyme inhibition. MPA significantly inhibited TNAP's phosphatase and pyrophosphatase activities at 10x and 100x physiological concentrations. In the presence of calcium [Ca(2+)] and [Mg(2+)] = 1 mM, MPA inhibited uncompetitively TNAP's phosphatase activity and inhibited noncompetitively its pyrophosphatase activity. CPPD crystal dissolution activity was also inhibited. Cysteine and MPA together in equimolar concentrations inhibited TNAP enzyme activities and CPPD crystal dissolution much more effectively than MPA or cysteine alone, reducing CPPD dissolution to 38% of controls at approximate physiologic inhibitor concentrations. Endogenous amino acids like cysteine and its derivative MPA have the capacity to inhibit TNAP activities at physiologic concentrations. Downregulation of their inhibiting concentration in the cartilage interstitial fluid environment may provide a therapeutic avenue to controlled dissolution of CPPD crystal deposition in tissues.

A novel spectrophotometric method for determination of kinetic constants of aldehyde oxidase using multivariate calibration method

Although phenanthridine has been frequently used as a specific substrate for the assessment of aldehyde oxidase activity, the use of this method is questionable due to a lower limit of detection and its validity for kinetic studies. In the present study, a novel sensitive multivariate calibration method based on partial least squares (PLS) has been developed for the measurement of aldehyde oxidase activity using phenanthridine as a substrate. Phenanthridine and phenanthridinone binary mixtures were prepared in a dynamic linear range of 0.1–30.0 μM and the absorption spectra of the solutions were recorded in the range of 210–280 nm in Sorenson's phosphate buffer (pH 7.0) containing EDTA (0.1 mM). The optimized PLS calibration model was used to calculate the concentration of each chemical in the prediction set. Hepatic rat aldehyde oxidase was partially purified and the initial oxidation rates of different concentrations of phenanthridine were calculated using the PLS method. The values were used for calculating Michaelis–Menten constants from a Lineweaver–Burk double reciprocal plot of initial velocity against the substrate concentration. The limits of detection for phenanthridine and phenanthridinone were found to be 0.04 ± 0.01 and 0.03 ± 0.01 μM (mean ± SD, n = 5), respectively. Using this method, the K m value for the oxidation of phenanthridine was calculated as 1.72 ± 0.09 μM (mean ± SD, n = 3). Thus, this study describes a novel spectrophotometric method that provides a suitable, sensitive and easily applicable means of measuring the kinetics of phenanthridine oxidation by aldehyde oxidase without the need for expensive instrumentation.

The c-Myc Target Gene Rcl (C6orf108) Encodes a Novel Enzyme, Deoxynucleoside 5′-monophosphate N-Glycosidase

RCL is a c-Myc target with tumorigenic potential. Genome annotation predicted that RCL belonged to the N-deoxyribosyltransferase family. However, its putative relationship to this class of enzymes did not lead to its precise biochemical function. The purified native or N-terminal His-tagged recombinant rat RCL protein expressed in Escherichia coli exhibits the same enzyme activity, deoxynucleoside 5'-monophosphate N-glycosidase, never before described. dGMP appears to be the best substrate. RCL opens a new route in the nucleotide catabolic pathways by cleaving the N-glycosidic bond of deoxynucleoside 5'-monophosphates to yield two reaction products, deoxyribose 5-phosphate and purine or pyrimidine base. Biochemical studies show marked differences in the terms of the structure and catalytic mechanism between RCL and of its closest enzyme family neighbor, N-deoxyribosyltransferase. The reaction products of this novel enzyme activity have been implicated in purine or pyrimidine salvage, glycolysis, and angiogenesis, and hence are all highly relevant for tumorigenesis.

C-terminal Tail Residue Arg1400 Enables NADPH to Regulate Electron Transfer in Neuronal Nitric-oxide Synthase

The neuronal nitric-oxide synthase (nNOS) flavoprotein domain (nNOSr) contains regulatory elements that repress its electron flux in the absence of bound calmodulin (CaM). The repression also requires bound NADP(H), but the mechanism is unclear. The crystal structure of a CaM-free nNOSr revealed an ionic interaction between Arg(1400) in the C-terminal tail regulatory element and the 2'-phosphate group of bound NADP(H). We tested the role of this interaction by substituting Ser and Glu for Arg(1400) in nNOSr and in the full-length nNOS enzyme. The CaM-free nNOSr mutants had cytochrome c reductase activities that were less repressed than in wild-type, and this effect could be mimicked in wild-type by using NADH instead of NADPH. The nNOSr mutants also had faster flavin reduction rates, greater apparent K(m) for NADPH, and greater rates of flavin auto-oxidation. Single-turnover cytochrome c reduction data linked these properties to an inability of NADP(H) to cause shielding of the FMN module in the CaM-free nNOSr mutants. The full-length nNOS mutants had no NO synthesis in the CaM-free state and had lower steady-state NO synthesis activities in the CaM-bound state compared with wild-type. However, the mutants had faster rates of ferric heme reduction and ferrous heme-NO complex formation. Slowing down heme reduction in R1400E nNOS with CaM analogues brought its NO synthesis activity back up to normal level. Our studies indicate that the Arg(1400)-2'-phosphate interaction is a means by which bound NADP(H) represses electron transfer into and out of CaM-free nNOSr. This interaction enables the C-terminal tail to regulate a conformational equilibrium of the FMN module that controls its electron transfer reactions in both the CaM-free and CaM-bound forms of nNOS.

A new enzyme model for enantioselective esterases based on molecularly imprinted polymers.

An efficient enzyme model exhibiting enantioselective esterase activity was prepared by using molecular imprinting techniques. The enantiomerically pure phosphonic monoesters 4 L and 5 L were synthesized as stable transition-state analogues. They were used as templates connected by stoichiometric noncovalent interactions to two equivalents of the amidinium binding site monomer 1. After polymerization and removal of the template, the polymers were efficient catalysts for the hydrolysis of certain nonactivated amino acid phenylesters (2 L, 2 D, 3 L, 3 D) depending on the template used. Imprinted catalyst IP4 (imprinted with 4 L) enhanced the hydrolysis of the corresponding substrate 2 L by a factor of 325 relative to that of a buffered solution. Relative to a control polymer containing the same functionalities, prepared without template 4 L, the enhancement was still about 80-fold, showing the highest imprinting effect up to now. In cross-selectivity experiments a strong substrate selectivity of higher than three was found despite small differences in the structure of the substrate and template. Plots of initial velocities of the hydrolysis versus substrate concentration showed typical Michaelis-Menten kinetics with saturation behavior. From these curves, the Michaelis constant K(M) and the catalytic constant k(cat) can be calculated. The enantioselectivity shown in these values is most interesting. The ratio of the catalytic efficiency k(cat)/K(M), between the hydrolysis of 2 L- and 2 D-substrate with IP4, is 1.65. This enantioselectivity derives from both selective binding of the substrate (K(M)L/K(M)D=0.82), and from selective formation of the transition state (k(cat)L/k(cat)D=1.36). Thus, these catalysts give good catalysis as well as high imprinting and substrate selectivity. Strong competitive inhibition is caused by the template used in imprinting. This behavior is also quite similar to the behavior of natural enzymes, for which these catalysts are good models.

Importance of product inhibition in the kinetics of the acylase hydrolysis reaction by differential stopped flow microcalorimetry

The hydrolysis of N-acetyl- l-methionine, N-acetylglycine, N-acetyl- l-phenylalanine, and N-acetyl- l-alanine at 298.35 K by porcine kidney acylase I (EC 3.5.1.14) was monitored by the heat released upon mixing of the substrate and enzyme in a differential stopped flow microcalorimeter. Values for the Michaelis constant ( K m) and the catalytic constant ( k cat) were determined from the progress of the reaction curve employing the integrated form of the Michaelis–Menten equation for each reaction mixture. When neglecting acetate product inhibition of the acylase, values for k cat were up to a factor of 2.3 larger than those values determined from reciprocal initial velocity–initial substrate concentration plots for at least four different reaction mixtures. In addition, values for K m were observed to increase linearly with an increase in the initial substrate concentration. When an acetate product inhibition constant of 600±31 M −1 , determined by isothermal titration calorimetry, was used in the progress curve analysis, values for K m and k cat were in closer agreement with their values determined from the reciprocal initial velocity versus initial substrate concentration plots. The reaction enthalpies, Δ r H(cal), which were determined from the integrated heat pulse per amount of substrate in the reaction mixture, ranged from −4.69±0.09 kJ mol −1 for N-acetyl- l-phenylalanine to −1.87±0.23 kJ mol −1 for N-acetyl- l-methionine.

Purine nucleoside phosphorylase from Cellulomonas sp.: physicochemical properties and binding of substrates determined by ligand-dependent enhancement of enzyme intrinsic fluorescence, and by protective effects of ligands on thermal inactivation of the enzyme

Purine nucleoside phosphorylase (PNP) from Cellulomonas sp., homotrimeric in the crystalline state, is also a trimer in solution. Other features of the enzyme are typical for “low molecular mass” PNPs, except for its unusual stability at pH 11. Purine bases, α- d-ribose-1-phosphate (R1P) and phosphate enhance the intrinsic fluorescence of Cellulomonas PNP, and hence form binary complexes and induce conformational changes of the protein that alter the microenvironment of tryptophan residue(s). The effect due to guanine (Gua) binding is much higher than those caused by other ligands, suggesting that the enzyme preferentially binds a fluorescent, most probably rare tautomeric anionic form of Gua, further shown by comparison of emission properties of the PNP/Gua complex with that of Gua anion and its N-methyl derivatives. Guanosine (Guo) and inosine (Ino) at 100 μM concentration show little and no effect, respectively, on enzyme intrinsic fluorescence, but their protective effect against thermal inactivation of the enzyme points to their forming weak binary complexes with PNP. Binding of Gua, hypoxanthine (Hx) and R1P to the trimeric enzyme is described by one dissociation constant, K d=0.46 μM for Gua, 3.0 μM for Hx, and 60 μM for R1P. The binding stoichiometry for Gua (and probably Hx) is three ligand molecules per enzyme trimer. Effects of phosphate on the enzyme intrinsic fluorescence are due not only to binding, but also to an increase in ionic strength, as shown by titration with KCl. When corrected for effects of ionic strength, titration data with phosphate are most consistent with one dissociation constant, K d=270 μM, but existence of a very weak binding site with K d>50 mM could not be unequivocally ruled out. Binding of Gua to the PNP/phosphate binary complex is weaker ( K d=1.7 μM) than to the free enzyme ( K d=0.46 μM), suggesting that phosphate helps release the purine base in the catalytic process of phosphorolysis. The results indicate that nonlinear kinetic plots of initial velocity, typical for PNPs, including Cellulomonas PNP, are not, as generally assumed, due to cooperative interaction between monomers forming the trimer, but to a more complex kinetic mechanism than hitherto considered.