Nonenzymatic peptide alpha-amidation. Implications for a novel enzyme mechanism

An abiotic system is described which chemically catalyzes the formation of less than Glu-His-Pro-NH2 (thyrotropin-releasing hormone) from less than Glu-His-Pro-amino acid in the presence of copper, ascorbate, and molecular oxygen. Evidence is presented to support the participation of hydroxyl and carbon radicals as reaction intermediates in the production of a peptide amide and an aldehyde or ketone. The characteristics of this model system closely mimic the characteristics of enzymatic peptide amidation, and an oxidative, free-radical mechanism for enzymatic peptide amidation is proposed as an alternative to the mechanism for enzymatic amidation offered by Bradbury et al. (Bradbury, A. F., Finnie, M. D. A., and Smyth, D. G. (1982) Nature 298, 686-688).     

A dynamical model for a co-operative enzyme.

Proteins partially immersed in the hydrophobic portion of a lipid bilayer interact by means of London-van der Waals non-bonding dispersion forces. Moreover, in certain organelles, enzymes are structured in a lattice or ordered matrix. These conditions may facilitate the establishment of long-range correlations between proteins. We studied the dynamical properties of a model for an enzyme endowed with a highly co-operative conformational transition between two reactive states. Two cases were considered, a closed system and an open system. In the closed system for different degrees of interaction among the proteins, it was found that for a substrate concentration greater than a certain threshold an abrupt change of enzymatic activity occurs. This biphasic behavior has been observed in the enzymatic activity of crystalline mitochondrial aspartate aminotransferase and for some other crystalline enzymes. In the analysis of the open system, for a specific input rate of the substrate, two different dynamics were found depending on the selected degree of interaction. For a certain value of a parameter phi, representing the degree of interaction among the reacting units, three steady states co-exist. This multiplicity confers excitable properties to the model. For larger values of phi, limit cycle type solutions were obtained. Thus, a sustained oscillatory product formation of the enzymatic reaction is observed. These results are compared with experimental observations of enzyme extracts detected by NMR.     

Prostaglandin H synthetase as a multisubstrate enzyme. Fluorimetric study of enzyme kinetics

The kinetics of a multisubstrate enzymatic reaction catalyzed by prostaglandin H synthase (PGH-synthase, EC 1.14.99.1) was studied, using homovanillic acid, a new electron donor for the given system. Homovanillic acid was shown to be a participant in a reaction with arachidonic acid/O2 stoichiometric ratios and is oxidized to a readily fluorescing product with an absorbance maximum (excitation) at 315 nm and fluorescence maximum at 425 nm. This allows for determination of the rate of enzymatic reaction with the sensitivity exceeding by one order of magnitude that of polarographic or spectrophotometric assays. Using fluorescent techniques, the dependence of the rate of PGH-synthase reaction on substrate (arachidonic acid, O2 and homovanillic acid) concentrations was studied, and the corresponding Km values were determined. The effect of Tween-20 and Lubrol PX concentrations on the reaction rate were examined. It was shown that with a decrease in the surfactant concentration the reaction rate increases.     

Neuroglobin and cytoglobin as potential enzyme or substrate

The possible enzymatic activities of neuro- and cytoglobin as well as their potential function as substrates in enzymatic reactions were studied. Neuro- and cytoglobin are found to show no appreciable superoxide dismutase, catalase, and peroxidase activities. However, the internal disulfide bond (CD7-D5) of human neuroglobin can be reduced by thioredoxin reductase. Furthermore, our in vivo and in vitro studies show that Escherichia coli cells contain an enzymatic reducing system that keeps the heme iron atom of neuroglobin in the Fe(2+) form in the presence of dioxygen despite the high autoxidation rate of the molecule. This reducing system needs a low-molecular-weight compound as co-factor. In vitro tests show that both NADH and NADPH can play this role. Furthermore, the reducing system is not specific for neuroglobin but allows the reduction of the ferric forms of other globins such as cytoglobin and myoglobin. A similar reducing system is present in eukaryotic tissue protein extracts.     

Regulation of enzyme activity in adsorptive enzyme systems. III. Interaction of pig muscle lactate dehydrogenase with F-actin

The binding of pig skeletal muscle lactate dehydrogenase by F-actin has been studied using the sedimentation method in 10 mM Tris-acetate buffer, pH 6.0 at 20 degrees C. Adsorption capacity of F-actin is equal to (1 +/- 0.1) . 10(-5) moles of lactate dehydrogenase per 1 g of actin. NADH decreases the affinity of F-actin with respect to lactate dehydrogenase. The binding of lactate dehydrogenase by F-actin in diminishing the rate of enzymatic reduction of alpha-ketoglutarate. The microscopic dissociation constant for the complex of the enzyme with F-actin which is estimated from the dependence of the enzymatic reaction rate of F-actin concentration at saturating NADH concentrations is equal (3.0 +2- 0.5) . 10(-7) M. It has been shown that the bound enzyme is characterized by the greater value of Km and the lower value of Vmax in comparison to the free enzyme.     

Vitamin K-dependent carboxylase. Requirements of the rat liver microsomal enzyme system.

Vitamin K is required in an enzymatic reaction which carboxylates glutamyl residues in a microsomal protein precursor of plasma prothrombin to form gamma-carboxyglutamic acid residues. The partial requirements of this microsomal, vitamin K-dependent carboxylase system have been determined. A requirement of the system for cytosolic factors appears to be due primarily to the presence of reduced pyridine nucleotides or a reduced pyridine nucleotide-generating system in the cytosol. The hydroquinone of vitamin K has been demonstrated to be the enzymatically active form of the vitamin. When vitamin K1 hydroquinone is added to the carboxylase system, no NAD(P)H is needed for maximum activity. The carboxylase activity is half-maximally stimulated by 0.25 mug of vitamin K1/ml in the presence of cytosolic components but requires at least 10 times as much vitamin when microsomes are incubated in a cytosol-free buffer. Menadione is inactive as a vitamin source in this system, and the carboxylase activity is inhibited by the 2-chloro analog of vitamin K1 and by Warfarin. The ATP analog, AMP-P(NH)P, inhibited the carboxylase activity, but a dependence on exogenous ATP or an ATP-generating system could not be demonstrated. Carboxylase activity was found to be dependent on an O2-containing gas phase, and upon the HCO3- concentration.     

Towards enzyme activated antiprostatic agents

A prodrug conjugate designed to undergo activation by enzymatic prostate specific antigen has been synthesized. The prodrug system undergoes activation with PSA or alpha-chymotrypsin, and shows selective cytotoxicity in a PSA secreting cell line.     

Electromagnetic induced kinetic effects on charged substrates in localized enzyme systems.

An analytical expression for the rate efficiency factor of planar localized enzyme systems is derived. The derivation takes into account the isothermal kinetic effect under the externally imposed perturbation of combined electrostatic and high frequency time-varying fields. The contribution of each individual field to the enzyme reaction is examined through the basic mechanism in which charged substrates interact with the specific perturbing field. The interaction mechanisms for the electrostatic and for the time-varying fields are found to be different. This difference regulates the different manners in which enzymatic reaction rates are altered. Enzymatic reactions under electrostatic perturbation can be retarded or enhanced depending on the field polarization. At sufficiently high field intensities the reaction rate may approach zero or approach a maximum value equal to the turnover number of the enzyme. Time-varying field perturbations, on the other hand, always enhance the enzymatic reactions if bunching effects are negligible. At sufficiently high field intensities, the reaction may approach a value equal to that of the free enzyme system. Several typical numerical examples on pure eletrostatic field perturbations, pure time-varying field perturbations, and combined field perturbations are also presented.     

Alpha complementation in the Cre recombinase enzyme

The Cre-loxP system is increasingly exploited for spatial and temporal gene inactivation. Here we present a novel approach to achieve this goal of selective gene inactivation. Following the model of alpha complementation in the beta-galactosidase enzyme, where the enzyme is split into independent polypeptides which are able to associate and maintain the enzymatic activity, we divided the Cre recombinase into two independent polypeptides (one containing the NH(2) terminus (alpha) and a second one containing the COOH-terminus (beta)). Individually, the two polypeptides have no detectable activity. However, when coexpressed the polypeptides are able to associate, giving rise to Cre enzymatic activity, which optimally is as high as 30% of that seen with wildtype Cre recombinase in vitro. We present this strategy as a modification of the traditional Cre-loxP system, which could be used to obtain a highly specific recombination pattern by expressing the two halves under the control of separate promoters.     

Kinetic amplification of enzyme discrimination.

The dependence of the accuracy of enzymatic systems on the mechanism of the catalyzed reaction is investigated, using a probabilistic approach. Certain mechanisms of reaction, involving a delay in one of the steps act as kinetic amplifiers of molecular discriminations. The relationship between our scheme for a delayed reaction [1] and Hopfield's scheme [i] is discussed.     

Metabolomics approach for enzyme discovery

The search for novel enzymes is an important but difficult task in functional genomics. Here, we present a systematic method based on in vitro assays in combination with metabolite profiling to discover novel enzymatic activities. A complex mixture of metabolites is incubated with purified candidate proteins and the reaction mixture is subsequently profiled by capillary electrophoresis electrospray ionization mass spectrometry (CE-MS). Specific changes in the metabolite composition can directly suggest the presence of an enzymatic activity while subsequent identification of the compounds whose level changed specifically can pinpoint the actual substrate(s) and product(s) of the reaction. We first evaluated the method using several Escherichia coli metabolic enzymes and then applied it to the functional screening of uncharacterized proteins. In this manner, YbhA and YbiV proteins were found to display both phosphotransferase and phosphatase activity toward different sugars/sugar phosphates. Our approach should be broadly applicable and useful for enzyme discovery in any system.     

Studies on prephenoloxidase-activating enzyme from cuticle of the silkworm Bombyx mori. II. Purification and characterization of the enzyme

Prephenoloxidase-activating enzyme has been purified approximately 4800-fold from cuticular extract of the silkworm, and the preparation seems to be homogeneous as judged by disc- and dodecylsulfate-polyacrylamide gel electrophoresis. By means of gel filtration through Sephadex G-100, it has been supposed that the enzyme exists as mono- and dimeric forms at slightly acidic pH, while a monomeric form is predominant under slightly alkaline condition. The molecular weight of the monomer was estimated to be 33,000–35,000 by dodecylsulfate-polyacrylamide gel electrophoresis and gel filtration.It has been demonstrated that ester substrates for trypsin, benzoyl-l-arginine ethyl ester and tosyl-l-arginine methyl ester, can be hydrolyzed by the purified enzyme. Several lines of evidence indicating that a single protein is involved in both activation and esterolytic reactions have been presented. Some enzymatic properties of the purified preparation as esterase have also been described.In connection to esterase activity of the purified enzyme, a mechanism of prephenoloxidase activation in the silkworm system has briefly been discussed.     

Fat fractal and multifractals for protein and enzyme surfaces.

The roughness and irregularity of the surfaces in the protein and enzyme are fractal features that may be characterized by fractal dimensions and mass exponents. The surface fractal dimensions calculated by the variation method are different from those obtained by other methods, since the former is applicable to the self-affine system. Thus the results reported here are reliable for the surfaces. However, the fat fractal and multifractal features of proteins and enzymes are studied by simulation. The surface mass exponents are regarded as another kind of scaling exponent, and the spectrum f(alpha) provides further detailed information about the surfaces of enzyme and protein. The applications of the spectrum f(alpha) to the enzymatic reactions is also discussed.     

Molecular mechanisms of systemic enzyme therapy

This work deals with the molecular mechanisms of system enzymotherapy agents action--both administrated perorally and intratestially sorbed enzymatic complexes. The basic of functional composite part of SET agents are supposed to be trypsin degraded forms.     

A microscopic model of enzyme kinetics.

Many in vivo enzymatic processes, such as those of the tissue factor pathway of blood coagulation, occur in environments with facilitated substrate delivery or enzymes bound to cellular or lipid surfaces, which are quite different from the ideal fluid environment for which the Michaelis-Menten equation was derived. To describe the kinetics of such reactions, we propose a microscopic model that focuses on the kinetics of a single-enzyme molecule. This model provides the foundation for macroscopic models of the system kinetics of reactions occurring in both ideal and nonideal environments. For ideal reaction systems, the corresponding macroscopic models thus derived are consistent with the Michaelis-Menten equation. It is shown that the apparent Km is in fact a function of the mechanism of substrate delivery and should be interpreted as the substrate level at which the enzyme vacancy time equals the residence time of ES-complexes; it is suggested that our microscopic model parameters characterize more accurately an enzyme and its catalytic efficiency than does the classical Km. This model can also be incorporated into computer simulations of more complex reactions as an alternative to explicit analytical formulation of a macroscopic model.     

Assay of proteolytic enzyme with a leucine-selective electrode.

A potentiometric method utilizing a leucine-selective membrane electrode has been developed for the assay of proteolytic enzyme. Subtilisin Carlsberg was used for the enzymatic digestion of casein in solution. Responses on the reaction media were linear on a semilog plot for active enzyme concentrations ranging from 0.6–60 μg/ml. The system uses commercially available components as well as simplified methods of solution handling without precipitation or filtration steps. This method offers significant advantages in terms of simplicity, speed, and cost over the traditional techniques.     

Human CD38, a surface receptor, an enzyme, an adhesion molecule and not a simple marker.

Human CD38 is a 45 kDa type II trans-membrane glycoprotein with a peculiar discontinuous pattern of expression in leukocytes, although evidence is accumulating of its quite widespread expression outside of the hematopoietic system. CD38 is a member of an emerging family of cytosolic and membrane-bound enzymes whose substrate is nicotinamide adenine dinucleotide (NAD), a coenzyme ubiquitously distributed in nature. CD38 is a multifunctional molecule able to exert not only an enzymatic activity but also to mobilize calcium, to transduce signals, to adhere to hyaluronan and to other ligands. Interaction with CD38 on various leukocyte subpopulation has profound though diverse effects on their life-span, however, the immunoregulatory activities seem to be independent of the enzymatic functions of the molecule.     

High pressure enzyme kinetics of dextransucrase.

The pressure dependence of enzymatic dextran formation has been observed up to 1000 at for several substrate concentrations. First order denaturation effects could be separated from the thermodynamic effects, which lead to a volume of 30.4 to 44.0 ccm per mole for the formation and -13.6ccm per mole for the activation of the enzyme-substrate complex. Denaturation depends on the substrate concentration. This leads to the conslusion that only the free enzyme is denatured, wheras the ES complex is stable.