Sticky Substrate Research Articles

Spectral and Kinetic Characterization of Intermediates in the Aromatization Reaction Catalyzed by NikD, an Unusual Amino Acid Oxidase

The flavoenzyme nikD, a 2-electron acceptor, catalyzes a remarkable aromatization of piperideine-2-carboxylate (P2C) to picolinate, an essential component of nikkomycin antibiotics. Steady-state kinetic data are indicative of a sequential mechanism where oxygen reacts with a reduced enzyme.dihydropicolinate (DHP) complex. The kinetics observed for complex formation with competitive inhibitors are consistent with a one-step binding mechanism. The anaerobic reaction with P2C involves three steps. The first step yields an enzyme.substrate charge transfer complex likely to contain the electron-rich P2C enamine. Calculated rates of formation and dissociation of the nikD.P2C complex are similar to those observed for the enzyme.1-cyclohexenoate complex. Formation of a reduced enzyme.DHP complex, (EH(2).DHP)(ini), occurs in a second step that exhibits a hyperbolic dependence on substrate concentration. The limiting rate of nikD reduction is at least 10-fold faster than the turnover rate observed with unlabeled or [4,4,5,5,6,6-D(6)]-P2C and exhibits a kinetic isotope effect (KIE = 6.4). The observed KIE on K(d apparent) (4.7) indicates that P2C is a sticky substrate. Formation of a final reduced species, (EH(2).DHP)(fin), occurs in a third step that is independent of P2C concentration and equal to the observed turnover rate. The observed KIE (3.3) indicates that the final step involves cleavage of at least one C-H bond. Tautomerization, followed by isomerization, of the initial DHP intermediate can produce an isomer that could be oxidized to picolinate in a reaction that satisfies known steric constraints of flavoenzyme reactions without the need to reposition a covalently tethered flavin or tightly bound intermediate.

Use of Prussian carp, <I>Carassius gibelio,</I> pituitaries in the artificial propagation of female common carp, <I>Cyprinus carpio</I>

Background. The usage of common carp pituitaries has been associated with very high costs, resulting from sac rificing the valuable broodstock. A close taxonomic proximity between the Common carp, Cyprinus carpio L., and Prussian carp, Carassius gibelio (Bloch, 1782), reflected in the presence of high levels of the homologous carp LH hormone in Prussian carp pituitaries, was recently demonstrated. This prompted the present authors to evaluate the Prussian carp pituitaries as a less expensive alternative to carp pituitaries, for inducing spawning in common carp. Materials and Methods. Prussian carp were fished in Lake Pamvotis (NW Greece) on April 4, 2007 and 204 pituitaries were extracted and processed. Accordingly, the prepared pituitary liquid extract was injected in 30 female common carp (experimental group) in the State Carp Hatchery in Psathotopi, (western Greece). Common carp pituitary extract was used for the hypophysation of female common carp in the control group ( n = 30). In both groups, standard hatchery procedures were followed concerning broodstock handling during the artificial propagation of common carp. Fertilized eggs were incubated in 7-L Zugar jars after removing the sticky substrate and hatching percentage was calculated. Results. Spawning success was 73.3%, total egg yield was 5.94 kg, total relative fecundity was 112.5 g · kg –1 of body weight (150.0 g · kg –1 of body weight based on fertile broodstock), and hatching percentage reached 85.2 %. Statistically, all the above performance values were not significantly different compared to the controls. Conclusion. Taking advantage of a natural resource practically unexploited, the use of Prussian carp pituitaries in the artificial propagation of common carp was equally effective to common carp pituitaries, contributing to lower running costs in the hatchery.

Kinetic and Structural Studies of Specific Protein−Protein Interactions in Substrate Catalysis by Cdc25B Phosphatase

Using a combination of steady-state and single-turnover kinetics, we probe substrate association, dissociation, and chemistry for the reaction of Cdc25B phosphatase with its Cdk2-pTpY/CycA protein substrate. The rate constant for substrate association for the wild-type enzyme is 1.3 x 10(6) M(-1) s(-1). The rate constant for dissociation is slow compared to the rate constant for phosphate transfer to form the phospho-enzyme intermediate (k2 = 1.1 s(-1)), making Cdk2-pTpY/CycA a sticky substrate. Compared to the wild type, all hotspot mutants of residues at the remote docking site that specifically affect catalysis with the protein substrate (Arg488, Arg492, and Tyr497 on Cdc25B and Asp206 on Cdk2) have greatly slowed rate constants of association (70- to 4500-fold), and some mutants have decreased k2 values compared to that of the wild type. Most dramatically, R492L, despite showing no significant changes in a crystal structure at 2.0 A resolution, has an approximately 100-fold decrease in k2 compared to that of wild-type Cdc25B. The active site C473S mutant binds tightly to and dissociates slowly from Cdk2-pTpY/CycA (Kd = 10 nM, k(off) = 0.01 s(-1)). In contrast, the C473D mutant, despite showing only localized perturbations in the active site at 1.6 A resolution, has a much weaker affinity and dissociates rapidly (Kd of 2 microM, k(off) > 2 s(-1)) from the protein substrate. Overall, we demonstrate that the association of Cdc25B with its Cdk2-pTpY/CycA substrate is governed to a significant extent by the interactions of the remote hotspot residues, whereas dissociation is governed by interactions at the active site.

Biomat-Related Lifestyles in the Precambrian

The strange biota of Neoproterozoic sea bottoms become more understandable if we assume that otherwise soft sediments were sealed by firm and erosion-resistant biomats. This allowed mat encrusters (vendobionts; trilobozoan and other sponges) to get attached to sandy bottoms, and molluscan mat scratchers to scrape off an algal film, as if they were living on rocks. Minute conical mat stickers (Cloudina) probably required a sticky substrate to become stabilized in upright position. Horizontal burrows are interpreted as the works of worm-like undermat miners. Only the latter lifestyle appears to go back to the Mesoproterozoic; the others emerged in Vendian times and virtually disappeared when matgrounds became restricted to hostile environments in the wake of the Cambrian ecological revolution.

PH studies on the mechanism of the pyridoxal phosphate-dependent dialkylglycine decarboxylase.

The pH dependence of the steady-state kinetic parameters for the dialkylglycine decarboxylase-catalyzed decarboxylation-dependent transamination between 2-aminoisobutyrate (AIB) and pyruvate is presented. The pH dependence of methylation and DTNB modification reactions, and spectroscopic properties, is used to augment the assignment of the kinetic pKa's to specific ionizations. The coincidence of pKa values (approximately 7.4) observed in kcat/KAIB, 1/KAIB, Kis for pyruvate, KPLP, and in absorbance and fluorescence titrations demonstrates that AIB is not a sticky substrate. It furthermore suggests that the decarboxylation step, or a conformational isomerization preceding it, limits the rate of the overall catalytic cycle. Coexisting, kinetically distinguishable conformers of DGD-PLP, originating from an alkali metal ion binding site, were previously demonstrated at pH 8.2 for DGD-PLP (Zhou, X., Toney, M. D. Biochemistry 37, 5761-5769). The pKa value of approximately 8.8 observed in kcat, kcat/KAIB, Kd for K+, spectrometric titrations, and the reaction of DGD-PLP with DTNB is tentatively assigned to the conformational change interconverting the two enzyme forms previously characterized. Three pKa's are observed in pH titrations of the DGD-PLP coenzyme absorbance. Individual spectra for the four ionization states are deconvoluted by fitting log-normal curves. All four ionization states have both ketoenamine and enolimine tautomers present. This and a review of spectral data in the literature lead to the conclusion that the pKa of approximately 7.4, which gives the largest spectral changes and controls kcat/KAIB, is not deprotonation of the aldimine nitrogen. Rather, it must be an active site residue whose ionization alters the ratio between ketoenamine and enolimine tautomers.

4-Oxalocrotonate tautomerase: pH dependence of catalysis and pKa values of active site residues.

The pH-rate profiles for the kinetic parameters of 4-oxalocrotonate tautomerase (4-OT) have been measured using 2-hydroxy-2,4-hexadiendioate (2a) and 2-hydroxy-2,4-pentadienoate (2b) as substrates. The pH dependences of log (kcat/Km) and of log kcat for the slow, nonsticky substrate 2b, which lacks a 6-carboxyl group, were bell-shaped with limiting slopes of unity on both sides of the pH optimum. For 2b, pKa values of 6.2 +/- 0.3 and 9.0 +/- 0.3 for the free enzyme (pKE) and 7.7 +/- 0.3 and 8.5 +/- 0.3 for the ES complex (pKES) were obtained. The pKE of 6.2 +/- 0.3 for 2b represents a true pKa for a basic group on the enzyme and is most likely Pro-1 on the basis of inhibition studies with the substrate-based affinity label 3-bromopyruvate (3-BP) [Stivers et al. (1996) Biochemistry 35, 803-813]. Accordingly, 15N NMR titration of the uniformly 15N-labeled enzyme showed that the pKa of the amino group of Pro-1 is 6.4 +/- 0.2, in reasonable agreement with those found by the effect of pH on kcat/Km for 2b (6.2 +/- 0.3) and on kinact/K1 for 3-BP (6.7 +/- 0.3), but three units lower than the pKa of the model compound proline amide (pKa = 9.4 +/- 0.2). The pKa values for the two histidine residues of 4-OT, which were measured by 1H NMR (His-6, pKa < or = 5; His-49, pKa = 5.2 +/- 0.2), are at least one pK unit lower than the pKE, excluding these residues as candidates for the general base. A plot of log (kcat/Km) vs pH for the 10(4)-fold more reactive, but sticky substrate 2a [(kcat/Km)max = 3.9 x 10(6) M-1 s-1] shows a limiting slope of two on the ascending limb indicating the ionization of two essential groups on the free enzyme and/or substrate. One of these groups, with a pKa value of 5.4, is reasonably assigned to the 6-carboxylate moiety of 2a (pKaCOOH = 5.4). This assignment is supported by the slope of unity for the ascending limb of log (kcat/Km) versus pH for 2b which lacks this group. Thus a negative charge at the 6-position is important for substrate binding and catalysis. The other group (pKa2 = 5.2) most likely represents a perturbed pKa for the general base Pro-1 (pKatrue = 6.4). The descending limb of log kcat/Km vs pH for 2a has a slope of unity and was fit to a single pKa3 = 10.3 +/- 0.2. The pH dependence of kcat for 2a gives pKa values for the ES complex (pKES) of 6.5 and 9.6. On the basis of these results, an isomerization mechanism involving general-base catalysis by a low pKa proline-1 and electrophilic catalysis by an as yet unidentified enzymic general-acid (pKa = 9.0) is proposed.

Energetics of thrombin-fibrinogen interaction.

The kinetic mechanism of thrombin-fibrinogen interaction has been elucidated by steady-state measurements of synthetic substrate hydrolysis by human alpha-thrombin in the presence of human fibrinogen used as a competitive inhibitor and sucrose used as a viscogenic agent. Sucrose greatly affects the FKm for thrombin-fibrinogen interaction, without altering the intrinsic properties of the system. Under conditions of pH 7.5 and 0.1 M NaCl, fibrinogen behaves like a sticky substrate for thrombin, with acylation being comparable to dissociation in the temperature range 20-37 degrees C. In the same temperature range, deacylation is much faster than acylation. The van't Hoff enthalpy of binding for thrombin-fibrinogen interaction is -24 +/- 3 kcal/mol and the entropy is -55 +/- 11 cal mol-1 deg-1. A chemical compensation effect is present in the binding of fibrinogen and synthetic amide substrates to thrombin, with the delta H and delta G values being linked through a linear relationship.

Mechanistic studies on thrombin catalysis

The kinetic mechanism of the cleavage of four p-nitroanilide (pNA) substrates by human alpha-thrombin has been investigated by using a number of steady-state kinetic techniques. Solvent isotope and viscosity effects were used to determine the stickiness of the substrates at the pH optimum of the reaction; a sticky substrate is defined as one that undergoes catalysis faster than it dissociates from the Michaelis complex. Whereas benzoyl-Arg-pNA could be classified as a nonsticky substrate, D-Phe-pipecolyl-Arg-pNA was very sticky. The other two substrates (tosyl-Gly-Pro-Arg-pNA and acetyl-D-Phe-pipecolyl-Arg-pNA) were slightly sticky. The pH profiles of kcat/Km were bell-shaped for all substrates. The pKa values determined from the pH dependence of kcat/Km for benzoyl-Arg-pNA were about 7.5 and 9.1. Similar pKa values were determined from the pH profiles of kcat/Km for tosyl-Gly-Pro-Arg-pNA and acetyl-D-Phe-pipecolyl-Arg-pNA and for the binding of the competitive inhibitor N alpha-dansyl-L-arginine-4-methylpiperidine amide. The groups responsible for the observed pKa values were proposed to be His57 and the alpha-amino group of Ile16. The temperature dependence of the pKa values was consistent with this assignment. The pKa values of 6.7 and 8.6 observed in the pH profile of kcat/Km for D-Phe-pipecolyl-Arg-pNA were displaced to lower values than those observed for the other substrates. The displacement of the acidic pKa value could be attributed to the stickiness of this substrate.(ABSTRACT TRUNCATED AT 250 WORDS)

Dihydrofolate reductase from Escherichia coli: the kinetic mechanism with NADPH and reduced acetylpyridine adenine dinucleotide phosphate as substrates.

Kinetic studies on the reaction catalyzed by dihydrofolate reductase from Escherichia coli have been undertaken with the aim of characterizing further the kinetic mechanism of the reaction. For this purpose, the kinetic properties of substrates were determined by measurement of (a) initial velocities over a wide range of substrate concentrations and (b) the stickiness of substrates in ternary enzyme complexes. Stickiness is defined as the rate at which a substrate reacts to give products relative to the rate at which that substrate dissociates. Stickiness was determined by varying the viscosity of reaction mixtures and the concentration of one substrate in the presence of a saturating concentration of the other substrate. The results indicate that NADPH is sticky in the enzyme-NADPH-dihydrofolate complex, while dihydrofolate is much less sticky in this complex. At higher concentrations, NADPH functions as an activator through the formation of an enzyme-NADPH-tetrahydrofolate from which tetrahydrofolate is released more rapidly than from an enzyme-tetrahydrofolate complex. Higher concentrations of dihydrofolate also cause enzyme activation, and it appears that this effect is due to the ability of dihydrofolate to displace tetrahydrofolate from a binary enzyme complex through the formation of a transitory enzyme-tetrahydrofolate-dihydrofolate complex. As NADPH and dihydrofolate function as activators and as NADPH behaves as a sticky substrate, the kinetic mechanism of the dihydrofolate reductase reaction with the natural substrates is steady-state random. By contrast with NADPH, reduced 3-acetylpyridine adenine dinucleotide phosphate exhibits only slight stickiness and does not function as an activator.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanism of the reaction catalyzed by dihydrofolate reductase from Escherichia coli: pH and deuterium isotope effects with NADPH as the variable substrate.

The variations with pH of the kinetic parameters and primary deuterium isotope effects for the reaction of NADPH with dihydrofolate reductase from Escherichia coli have been determined. The aims of the investigations were to elucidate the chemical mechanism of the reaction and to obtain information about the location of the rate-limiting steps. The V and V/KNADPH profiles indicate that a single ionizing group at the active center of the enzyme must be protonated for catalysis, whereas the Ki profiles show that the binding of NADPH to the free enzyme and of ATP-ribose to the enzyme-dihydrofolate complex is pH independent. From the results of deuterium isotope effects on V/KNADPH, it is concluded that NADPH behaves as a sticky substrate. It is this stickiness that raises artificially the intrinsic pK value of 6.4 for the Asp-27 residue of the enzyme-dihydrofolate complex [Howell, E. E., Villafranca, J. E., Warren, M. S., Oatley, S. J., & Kraut, J. (1986) Science (Washington, D.C.) 231, 1123] to an observed value of 8.9. Thus, the binary enzyme complex is largely protonated at neutral pH. The elevation of the intrinsic pK value of 6.4 for the ternary enzyme-NADPH-dihydrofolate complex to 8.5 is not due to the kinetic effects of substrates. Rather, it is the consequence of the lower, pH-independent rate of product release and the faster pH-dependent catalytic step. At neutral pH, the proportion of enzyme present as a protonated ternary enzyme-substrate complex is sufficient to keep catalysis faster than product release.(ABSTRACT TRUNCATED AT 250 WORDS)

PH studies on the chemical mechanism of rabbit muscle pyruvate kinase. 2. Physiological substrates and phosphoenol-alpha-ketobutyrate.

pH profiles have been determined for the reactions catalyzed by pyruvate kinase between pyruvate and MgATP and between phosphoenolpyruvate and MgADP. V, V/KMgATP, and V/Kpyruvate all decrease below a pK of 8.3 and above one of 9.2. The group with pK = 8.3 is probably a lysine that removes the proton from pyruvate during enolization, while the pK of 9.2 is that of water coordinated to enzyme-bound Mg2+. The fact that this pK shows in all three pH profiles shows that pyruvate forms a predominantly second sphere complex and cannot replace hydroxide to form the inner sphere complex that results in enolization and subsequent phosphorylation. On the basis of the displacement of the pK of the acid-base catalytic group in its V/K profile, phosphoenolpyruvate is a sticky substrate, reacting to give pyruvate approximately 5 times faster than it dissociates. The V/K profile for the slow substrate phosphoenol-alpha-ketobutyrate shows the pK of 8.3 for the acid-base catalytic group in its correct position, but this group must be protonated so that it can donate a proton to the intermediate enolate following phosphoryl transfer. The secondary phosphate pK of the substrate is seen in this V/K profile as well as in the pKi profile for phosphoglycolate (but not in those for glycolate O-sulfate or oxalate), showing a preference for the trianion for binding. The chemical mechanism with the natural substrates thus appears to involve phosphoryl transfer between MgADP and a Mg2+-bound enolate with metal coordination of the enolate serving to make it a good leaving group.

Airborne particulate size distribution measurements in nickel smelter plumes

This paper reports the results of particle sizing measurements in the plume from two smelters at Sudbury, Ontario (INCO and Falconbridge) which were studied in 1979 and 1980. Andersen impactors, mounted on a Hughes 500C helicopter, were used. Due to bounce-off problems, it was found necessary to use sticky substrates for the coarse particles. In both the INCO and Falconbridge smelters, constituents could be classified as coarse particles having mass median diameter equal to or greater than 2.5 μm (including Fe, Cu, Ni, Al and Mn) and fine particles having mass median diameter less than 2.5 μm (including SO 4Pb, Zn, Cd and As). Most of these distributions were not log-normal, but displayed bimodal or multi-modal characteristics.

The Influence of Host Plants on Parasitism of Eggs of the Tobacco Hornworm12

Eggs of Manduca sexta (Johannson) were parasitized by Tetenomus sphingis (Ashmead), Trichogramma minutum Riley, and Anastatus sp. on solanaceous hosts other than tobacco in North Carolina. Although low and variable during midsummer, parasitism rose to 48% of naturally occurring eggs in September on Jimson-weed, Datura stramonium L. At this time 75% of the parasitized eggs yielded Telenomus sphingis and 25% Trichogramma minutum . Comparable samples of eggs from tobacco were not parasitized. In the laboratory, T. minutum failed to parasitize eggs on fresh tobacco foliage but readily attacked eggs on other substrates. The tiny parasites became stuck to the tobacco leaf in the gummy exudate of the trichomes. Telenomus sphingis , being larger than Trichogramma minutum , was able to cope with the sticky substrate to some extent, but Its movements on tobacco were inhibited and it parasitized significantly fewer eggs on tobacco than on any other substrate.