Extracellular enzyme localization during interspecific fungal interactions

Abstract Confronted colonies of Phlebia radiata, P. rufa, Coriolus versicolor, Stereum hirsutum, Phanerochaete velutina and Hypholoma fasciculare showed spatially and temporally heterogeneous patterns of loccase-α-naphthol and peroxidase activities. These activities were coincident in axenic cultures. but were not always so during interaction. Confrontation between species resulted in induction of phenoloxidase activities, even within coenocytic colony regions of Phlebia species which were normally void of such activities in axenic culture. These events resulted in restriction of C. versicolor growth during interaction with P. rufa.     

Glycolytic enzyme interactions with tubulin and microtubules

Interactions of the glycolytic enzymes glucose-6-phosphate isomerase, aldolase, glyceraldehyde-3-phosphate dehydrogenase, triose-phosphate isomerase, enolase, phosphoglycerate mutase, phosphoglycerate kinase, pyruvate kinase, lactate dehydrogenase type-M, and lactate dehydrogenase type-H with tubulin and microtubules were studied. Lactate dehydrogenase type-M, pyruvate kinase, glyceraldehyde-3-phosphate dehydrogenase, and aldolase demonstrated the greatest amount of co-pelleting with microtubules. The presence of 7% poly(ethylene glycol) increased co-pelleting of the latter four enzymes and two other enzymes, glucose-6-phosphate isomerase, and phosphoglycerate kinase with microtubules. Interactions also were characterized by fluorescence anisotropy. Since the KD values of glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase and lactate dehydrogenase for tubulin and microtubules were all found to be between 1 and 4 microM, which is in the range of enzyme concentration in cells, these enzymes are probably bound to microtubules in vivo. These observations indicate that interactions of cytosolic proteins, such as the glycolytic enzymes, with cytoskeletal components, such as microtubules, may play a structural role in the formation of the microtrabecular lattice.     

Studying non-covalent enzyme carbohydrate interactions by STD NMR

Saturation transfer difference NMR spectroscopy is used to study non-covalent interactions between four different glycostructure transforming enzymes and selected substrates and products. Resulting binding patterns represent a molecular basis of specific binding between ligands and biocatalysts. Substrate and product binding to Aspergillus fumigatus glycosidase and to Candida tenuis xylose reductase are determined under binding-only conditions. Measurement of STD effects in substrates and products over the course of enzymatic conversion provides additional information about ligand binding during reaction. Influences of co-substrates and co-enzymes in substrate binding are determined for Schizophyllum commune trehalose phosphorylase and C. tenuis xylose reductase, respectively. Differences between ligand binding to wild type enzyme and a corresponding mutant enzyme are shown for Corynebacterium callunae starch phosphorylase and its His-334-->Gly mutant. The resulting binding patterns are discussed with respect to the possibility that ligands do not only bind in the productive mode.     

Plasma protein binding and endothelial enzyme interactions in the lung

The influence of plasma albumin binding of the synthetic angiotensin-converting enzyme (ACE) substrate [3H]benzoyl-phenylalanyl-alanyl-proline (BPAP) on BPAP hydrolysis by pulmonary endothelial ACE was studied in isolated rabbit lungs perfused with a salt solution containing either 5% bovine serum albumin (BSA) or 5% dextran. The single-pass indicator-dilution method was used to measure the fraction (M) of [3H]BPAP hydrolyzed. Lung M was greater with albumin-free perfusate than when BSA was present. M decreased as the time (ti) that the BPAP was in contact with the BSA before reaching the lung was increased, suggesting that some BSA binding sites for BPAP were not in equilibrium during bolus transit through the lungs. The M vs. ti data were correlated using a model incorporating both rapid and slow binding kinetics of BPAP and BSA. For the slow BPAP-BSA interaction, the dissociation rate constant was approximately 0.015 s-1, and the fraction of the BPAP bound to these slowly equilibrating sites at equilibrium was approximately 22%. The results indicate that transient plasma protein binding kinetics can affect lung BPAP hydrolysis.     

Localized structural effects of electrostatic interactions in a thermostable enzyme

The sequence and resolved structure of thermotrophic isopropylmalate dehydrogenase (IPMDH) and a related protein, mesotrophic isocitrate dehydrogenase (IDH), were compared emphasizing clusters of charged residues identified from X-ray crystallographic studies (Wallon, G., Kryger, G., Lovett, S. T., Oshima, T., Ringe, D., and Petsko, G. A. (1997) J. Mol. Biol. 266, 1016-1031). Mesotrophic isocitrate dehydrogenase was used for comparison because crystallographic data for a mesotrophic form of IPMDH was not available in the database. The structural features in the region of these clusters were compared and localized conformational differences were found in the thermotroph compared to the mesotroph. Because the overall topology of the two proteins is similar, it was concluded that these localized structural differences induced by electrostatic interactions between charged residues in the thermotrophic enzyme were responsible for the enhanced thermal stability of proteins from thermotroph.     

Binding and conformational analysis of phosphoramidate-restriction enzyme interactions

Phosphoramidates are modified deoxyoligonucleotides that feature nitrogen in place of the 3'-oxygen of a phosphodiester linkage. Noted for stability against nuclease activity, these linkages are of both mechanistic and therapeutic interest. While a number of studies characterizing the properties of oligonucleotides composed entirely of phosphoramidate linkages have been published, little is known about how singly substituted phosphoramidate substitutions affect the thermodynamics and structure of protein-oligonucleotide interactions. We chose to investigate these interactions with PvuII endonuclease, the DNA binding behavior of which is well-characterized. Oligonucleotide duplexes containing a phosphoramidate substitution at the scissile phosphates were resistant to cleavage by the enzyme, even after extended incubations. However, the enzyme was able to cleave the native strand in a native:phosphoramidate heteroduplex at a rate comparable to that observed with the native substrate. Ca(II)-stimulated PvuII binding for a phosphoramidate-substituted oligonucleotide is comparable to that of the native duplex (K(d) approximately 200 pM). K(d) values obtained in the presence of Mg(II) are somewhat weaker (K(d) approximately 10 nM). Under metal-free conditions, the enzyme exhibited a remarkable approximately 50-fold greater affinity for the modified oligonucleotide relative to the native substrate (5 vs 240 nM). While (31)P NMR spectra indicate increased chemical shift dispersion in the free phosphoramidate duplex, the spectrum of the enzyme-bound duplex is similar to that of the native duplex. (1)H-(15)N HSQC analysis indicates that enzyme conformations in the presence of these oligonucleotides are also comparable. The tight binding of the phosphoramidate duplex under metal-free conditions and its resistance to cleavage are attributed to local conformational adjustments propagating from the O-->N substitution.     

Computer simulations of glycolytic enzyme interactions with F-actin

Muscle actin and fructose-1,6-bisphosphate aldolase (aldolase) were chemically crosslinked to produce an 80 kDa product representing one subunit of aldolase linked to one subunit of actin. Hydroxylamine digestion of the crosslinked product resulted in two 40.5 kDa fragments, one that was aldolase linked to the 12 N-terminal residues of actin. Brownian dynamics simulations of muscle aldolase and GAPDH with F-actin (muscle, yeast, and various mutants) estimated the association free energy. Mutations of residues 1-4 of muscle actin to Ala individually or two in combination of the first four residues reduced the estimated binding free energy. Simulations showed that muscle aldolase binds with the same affinity to the yeast actin as to the double mutated muscle actin; these mutations make the N-terminal of muscle actin identical to yeast, supporting the conclusion that the actin N-terminus participates in binding. Because the depth of free energy wells for yeast and the double mutants is less than for native rabbit actin, the simulations support experimental findings that muscle aldolase and GAPDH have a higher affinity for muscle actin than for yeast actin. Furthermore, Brownian dynamics revealed that the lower affinity of yeast actin for aldolase and GAPDH compared to muscle actin, was directly related to the acidic residues at the N-terminus of actin.     

Linear regression analysis for enzyme kinetic studies in toxicant interactions

A new method based on linear regression of per cent modulation in enzyme activity over substrate concentration is proposed to give an interpretative base to the influence of more than one modulator/inhibitor/toxicant, a situation commonly met with in toxicological studies-toxicant interactions. Advantages of this method over the classical kinetic methods are discussed in detail using brain Mg2+ ATPase activity of the fish Tilapia mossambica under in vitro regimes of hexachlorocyclohexane and malathion.     

Ca2+-surfactant interactions affect enzyme stability in detergent solutions

Detergent proteases and amylases generally bind Ca(2+) ions. These bound ions enhance enzyme stability, reducing the rates of degradative reactions such as unfolding and proteolysis. Thus, surfactant aggregates, such as micelles, affect protease and amylase stability indirectly, by competing with the enzymes for Ca(2+) ions. Dissociation constants for Ca(2+) interactions with anionic surfactant micelles are in the 10(-3) to 10(-2) M range. These interactions are weak relative to enzyme-Ca(2+) interactions (K(d) of order 10(-6) M). However, surfactant is typically present at much higher concentration than enzyme, and it is the Ca(2+)-micelle equilibrium that largely determines the amount of free Ca(2+) available for binding to enzymes. The problem of surfactant-mediated Ca(2+) removal from enzymes can be avoided by adding calcium to a detergent formulation in an amount such that the concentration of free Ca(2+) is around 10(-5)M.     

Dynamic interactions between enzyme activity and the microstructured environment

A new approach for the study of an enzyme's relationship with its own reaction medium has been developed. One technique of micellar enzymology is the use of pseudohomogeneous systems composed of surfactant/water/organic solvent. In such systems, the physicochemical properties and textures of the medium depend on the relative ratios of the different components. Enzymes are catalytically active in such systems and up to the present have been studied in different microenvironments, such as micelles, microemulsions and lyotropic liquid crystals. Our purpose was to develop a system in which the enzyme could, by its activity, modify one of the components in such a way that the relative ratios among them changed sufficiently to produce a transition from one phase domain to another. The three components, water (or glucose in water), octanol and octyl-beta-D-glucoside, form a classical ternary water/oil/surfactant system. The relevant phase diagram shows different macroheterogeneous phases and microstructured domains. The enzyme beta-D-glucosidase hydrolyses octyl-beta-D-glucoside to form glucose and octanol. The enzyme was found to change the relative ratios of water (or glucose in water), octanol and octyl-beta-D-glucoside in such a manner that the physicochemical structure of the medium was modified. At the beginning of the reaction beta-D-glucosidase was present in a micellar solution of octyl-beta-D-glucoside in water. As the enzymatic reaction proceeded, the medium became biphasic. One of the two phases was the micellar solution of octyl beta-D-glucoside in water, while the other phase was either a microemulsion or a liquid crystalline phase. In addition the enzyme, through its catalytic activity, was able to modify the physiocochemical properties of the reaction medium.     

Lectin-carbohydrate interactions studied by a competitive enzyme inhibition assay

The principle of competitive binding assay in combination with an immobilized lectin (concanavalin A), in close proximity to an oxygen sensor, has been used to quantify carbohydrates and to determine association constants for lectin-carbohydrate interactions. Methyl α-d-mannopyranoside was determined down to 0.5 μg/ml. K_a (maltose) and K_a (maltotriose) was found to be 2.1 × 10³ and 1.7 × 10³m^?1, respectively, which are comparable to values quoted in the literature of approximately 2.8 × 10³m^?1 for both maltose and maltotriose. Furthermore, the estimation of the bonus effect, due to multipoint attachment, for a low-molecular-weight dextran is discussed.     

Glycolytic enzyme interactions with yeast and skeletal muscle F-actin

Interaction of glycolytic enzymes with F-actin is suggested to be a mechanism for compartmentation of the glycolytic pathway. Earlier work demonstrates that muscle F-actin strongly binds glycolytic enzymes, allowing for the general conclusion that "actin binds enzymes", which may be a generalized phenomenon. By taking actin from a lower form, such as yeast, which is more deviant from muscle actin than other higher animal forms, the generality of glycolytic enzyme interactions with actin and the cytoskeleton can be tested and compared with higher eukaryotes, e.g., rabbit muscle. Cosedimentation of rabbit skeletal muscle and yeast F-actin with muscle fructose-1,6-bisphosphate aldolase (aldolase) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) followed by Scatchard analysis revealed a biphasic binding, indicating high- and low-affinity domains. Muscle aldolase and GAPDH showed low-affinity for binding yeast F-actin, presumably because of fewer acidic residues at the N-terminus of yeast actin; this difference in affinity is also seen in Brownian dynamics computer simulations. Yeast GAPDH and aldolase showed low-affinity binding to yeast actin, which suggests that actin-glycolytic enzyme interactions may also occur in yeast although with lower affinity than in higher eukaryotes. The cosedimentation results were supported by viscometry results that revealed significant cross-linking at lower concentrations of rabbit muscle enzymes than yeast enzymes. Brownian dynamics simulations of yeast and muscle aldolase and GAPDH with yeast and muscle actin compared the relative association free energy. Yeast aldolase did not specifically bind to either yeast or muscle actin. Yeast GAPDH did bind to yeast actin although with a much lower affinity than when binding muscle actin. The binding of yeast enzymes to yeast actin was much less site specific and showed much lower affinities than in the case with muscle enzymes and muscle actin.     

Subunit interactions in enzyme transition states--antagonism between substrate binding and reaction rate

The principles of structural kinetics as applied to polymeric enzymes have been reinvestigated in order to take account of the probable existence of subunit interactions in the enzyme transition states. On the basis of simple and plausible postulates, structural rate equations have been derived for dimeric enzymes and compared to substrate binding isotherms. It then becomes possible to understand how subunit interactions affect substrate affinity and enzyme reaction rate. There exists an antagonism between substrate binding to the enzyme and the steady state rate of product appearance. If subunit interactions increase the rate of product appearance, they decrease the fractional saturation of the enzyme by the substrate. Alternatively, if they decrease the reaction velocity they increase the fractional saturation. This seemingly paradoxical effect is the direct consequence of subunit interactions occurring in both the ground and the transition states.     

Protein phosphorylation in amyloplasts regulates starch branching enzyme activity and protein-protein interactions

Protein phosphorylation in amyloplasts and chloroplasts of Triticum aestivum (wheat) was investigated after the incubation of intact plastids with gamma-(32)P-ATP. Among the soluble phosphoproteins detected in plastids, three forms of starch branching enzyme (SBE) were phosphorylated in amyloplasts (SBEI, SBEIIa, and SBEIIb), and both forms of SBE in chloroplasts (SBEI and SBEIIa) were shown to be phosphorylated after sequencing of the immunoprecipitated (32)P-labeled phosphoproteins using quadrupole-orthogonal acceleration time of flight mass spectrometry. Phosphoamino acid analysis of the phosphorylated SBE forms indicated that the proteins are all phosphorylated on Ser residues. Analysis of starch granule-associated phosphoproteins after incubation of intact amyloplasts with gamma-(32)P-ATP indicated that the granule-associated forms of SBEII and two granule-associated forms of starch synthase (SS) are phosphorylated, including SSIIa. Measurement of SBE activity in amyloplasts and chloroplasts showed that phosphorylation activated SBEIIa (and SBEIIb in amyloplasts), whereas dephosphorylation using alkaline phosphatase reduced the catalytic activity of both enzymes. Phosphorylation and dephosphorylation had no effect on the measurable activity of SBEI in amyloplasts and chloroplasts, and the activities of both granule-bound forms of SBEII in amyloplasts were unaffected by dephosphorylation. Immunoprecipitation experiments using peptide-specific anti-SBE antibodies showed that SBEIIb and starch phosphorylase each coimmunoprecipitated with SBEI in a phosphorylation-dependent manner, suggesting that these enzymes may form protein complexes within the amyloplast in vivo. Conversely, dephosphorylation of immunoprecipitated protein complex led to its disassembly. This article reports direct evidence that enzymes of starch metabolism (amylopectin synthesis) are regulated by protein phosphorylation and indicate a wider role for protein phosphorylation and protein-protein interactions in the control of starch anabolism and catabolism.