Expression Of Catalytic Activity Research Articles

Shiga toxins: from structure and mechanism to applications.

Shiga toxins are a group of type 2 ribosome-inactivating proteins (RIPs) produced in several types of bacteria. The toxins possess an AB5 structure, which comprises a catalytic A chain with N-glycosidase activity, and five identical B chains and recognize and bind to the target cells with specific carbohydrate moieties. In humans, the major molecular target which recognizes the Shiga toxins is the Gb3 receptor, which is mainly expressed on the cell surface of endothelial cells of the intestine, kidney, and the brain. This causes these organs to be susceptible to the toxicity of Shiga toxins. When a person is infected by Shiga toxin-producing bacteria, the toxin is produced in the gut, translocated to the circulatory system, and carried to the target cells. Toxicity of the toxin causes inflammatory responses and severe cell damages in the intestine, kidneys, and brain, bringing about the hemolytic uremic syndrome (HUS), which can be fatal. The Shiga toxin requires a couple of steps to exert its toxicity to the target cells. After binding with the target cell surface receptor, the toxin requires a complicated process to be transported into the cytosol of the cell before it can approach the ribosomes. The mechanisms for the interactions of the toxin with the cells are described in this review. The consequences of the toxin on the cells are also discussed. It gives an overview of the steps for the toxin to be produced and transported, expression of catalytic activity, and the effects of the toxin on the target cells, as well as effects on the human body.

Expression of Allosteric Linkage between the Sodium Ion Binding Site and Exosite I of Thrombin during Prothrombin Activation

The specificity of thrombin for procoagulant and anticoagulant substrates is regulated allosterically by Na+. Ordered cleavage of prothrombin (ProT) at Arg320 by the prothrombinase complex generates proteolytically active, meizothrombin (MzT), followed by cleavage at Arg271 to produce thrombin and fragment 1.2. The alternative pathway of initial cleavage at Arg271 produces the inactive zymogen form, the prethrombin 2 (Pre 2).fragment 1.2 complex, which is cleaved subsequently at Arg320. Cleavage at Arg320 of ProT or prethrombin 1 (Pre 1) activates the catalytic site and the precursor form of exosite I (proexosite I). To determine the pathway of expression of Na+-(pro)exosite I linkage during ProT activation, the effects of Na+ on the affinity of fluorescein-labeled hirudin-(54-65) ([5F]Hir-(54-65)(SO-3)) for the zymogens, ProT, Pre 1, and Pre 2, and for the proteinases, MzT and MzT-desfragment 1 (MzT(-F1)) were quantitated. The zymogens showed no significant linkage between proexosite I and Na+, whereas cleavage at Arg320 caused the affinities of MzT and MzT(-F1) for [5F]Hir-(54-65)(SO-3) to be enhanced by Na+ 8- to 10-fold and 5- to 6-fold, respectively. MzT and MzT(-F1) showed kinetically different mechanisms of Na+ enhancement of chromogenic substrate hydrolysis. The results demonstrate for the first time that MzT is regulated allosterically by Na+. The results suggest that the distinctive procoagulant substrate specificity of MzT, in activating factor V and factor VIII on membranes, and the anticoagulant, membrane-modulated activation of protein C by MzT bound to thrombomodulin are regulated by Na+-induced allosteric transition. Further, the Na+ enhancement in MzT activity and exosite I affinity may function in directing the sequential ProT activation pathway by accelerating thrombin formation from the MzT fast form.

Role of Prothrombin Fragment 1 in the Pathway of Regulatory Exosite I Formation during Conversion of Human Prothrombin to Thrombin

Prothrombin (Pro) activation by factor Xa generates the thrombin catalytic site and exosites I and II. The role of fragment 1 (F1) in the pathway of exosite I expression during Pro activation was characterized in equilibrium binding studies using hirudin(54-65) labeled with 6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoate ([NBD]Hir(54-65)(SO3-)) or 5-(carboxy)fluorescein ([5F]Hir(54-65)(SO3-)). [NBD]Hir(54-65)(SO3-) distinguished exosite I environments on Pro, prethrombin 1 (Pre 1), and prethrombin 2 (Pre 2) but bound with the same affinities as [5F]Hir(54-65)(SO3-). Conversion of Pro to Pre 1 caused a 7-fold increase in affinity for the peptides. Conversely, fragment 1.2 (F1.2) decreased the affinity of Pre 2 for [5F]Hir(54-65)(SO3-) by 3-fold. This was correlated with a 16-fold increased affinity of F1.2 for Pre 2 in comparison to thrombin, demonstrating an enhancing effect of F1 on F1.2 binding. The active intermediate, meizothrombin, demonstrated a 50- to 220-fold increase in exosite affinity. Free thrombin and thrombin.F1.2 complex bound [5F]Hir(54-65)(SO3-) with indistinguishable affinity, indicating that the effect of F1 on peptide binding was eliminated upon expression of catalytic activity and exosite I. The results demonstrate a new zymogen-specific role for F1 in modulating the affinity of ligands for exosite I. This may reflect a direct interaction between the F1 and Pre 2 domains in Pro that is lost upon folding of the zymogen activation domain. The effect of F1 on (pro)exosite I and the role of (pro)exosite I in factor Va-dependent substrate recognition suggest that the Pro activation pathway may be regulated by (pro)exosite I interactions with factor Va.

Cyclooxygenase activity is regulated by the heme oxygenase system in microvessel endothelial cells.

Cyclooxygenase(s) catalyzes the oxygenation and peroxidation of arachidonic acid to prostaglandin endoperoxide H2, the immediate precursor of prostaglandins and thromboxane. Two COX isoforms encoded by two related genes have been identified; COX-1 is constitutively expressed and is considered to generate prostaglandins for normal physiological functions whereas COX-2 is, in most tissues, an inducible enzyme expressing rapidly and transiently in response to a variety of stimuli [1]. Both COX isoforms are hemeproteins [[2]. Heme binds to the COX apoenzyme with a stoichiometry of approximately one heme molecule per each subunit [[3]]. It is well documented that the heme prosthetic group of COX is essential for the expression of catalytic activity [[3]]. Accordingly, the possibility arises that variations in the cellular levels of heme impact on the amount of catalytically active COX present in cells.

Inhibition of the insulin receptor kinase phosphorylation by nitric oxide: functional and structural aspects.

Previous studies on cultured skeletal muscle cells have indicated that the insulin-induced expression of GLUT4 transporter protein is inhibited by nitric oxide (NO). Therefore, we determined the effect of NO on the insulin-induced autophosphorylation of the insulin receptor kinase (IRK), i.e., the first step in the insulin-mediated signal transduction pathway. The experiments showed that the insulin-induced autophosphorylation of the insulin receptor beta-chain is strongly inhibited by the NO donors 1,1-diethyl-2-hydroxy-2-nitrosohydrazine (DEA-NO) or S-nitroso-N-acetylpenicillamine (SNAP). The inhibitory effect was ameliorated in cells depleted of glutathione (GSH), suggesting the possibility that S-nitroso-glutathione may operate as an intermediate NO donor. Complementary experiments with different Cys --> Ala mutant proteins showed, surprisingly, that all mutant proteins were inhibited by DEA-NO. Three-dimensional models of the nonphosphorylated IR beta-chain nitrosylated at the accessible cysteine residues 1056, 1138, 1234, or 1245 revealed that derivatization of any of these four cysteine residues leads essentially to the same structural changes of the IRK domain. These changes involve a movement of the amino-terminal lobe against the carboxy-terminal lobe in a direction opposite to the direction of the "lobe closure" that was previously proposed to facilitate the accessibility for ATP and the expression of catalytic activity. Our findings suggest that the occurrence of several functionally relevant cysteine residues in distinct regions of the IRK protein increases the probability of regulatory redox interactions and thus the redox sensitivity of the IRK.

Molecular cloning of acetyl coenzyme a: Deacetylcephalosporin C o-acetyltransferase cDNA from Acremonium chrysogenum: Sequence and expression of catalytic activity in yeast

Acetyl CoA: deacetylcephalosporin C o-acetyltransferase(DCPC-ATF) catalyses the final step in the biosynthesis of cephalosporin C, the conversion of deacetylcephalosporin C to cephalosporin C. A cDNA encoding DCPC-ATF has been isolated from a cDNA library of a cephalosporin C producing fungus Acremonium chrysogenum using oligonucleotide probes based on N-terminal amino acid sequences of the enzyme. The cDNA contains a single large open reading frame for a putative precursor consisting of 12 amino acid(AA) leader peptide of unknown function, 274 AA large subunit and 126 AA small subunit at the carboxyl end. The cDNA was expressed in yeast exhibiting a functional DCPC-ATF activity. It was also indicated that the leader peptide was not essential for expression of the enzyme activity. The primary structure of DCPC-ATF shows significant homology with those of acetyl CoA: homoserine o-acetyltransferase in Saccharomyces cerevisiae and Ascobolas immersus.

Molecular cloning of mouse acid β-galactosidase cDNA: Sequence, expression of catalytic activity and comparison with the human enzyme

A full-length cDNA coding for mouse lysosomal acid β-galatosidase has been isolated on the basis of homology with the human gene. Catalytic activity toward 4-methylumbelliferyl β-D-galactoside in the COS-1 cell expression system provided positive proof for its authenticity. The sequence analysis showed that the degree of similarity between the human and mouse enzymes was approximately 70% in the nucleotide sequence and nearly 80% in the amino acid sequence. The deduced primary amino acid sequences of the enzymes from the two species indicated that, of the seven possible N-glycosylation sites in the human enzyme, five are conserved in the mouse enzyme. Three additional possible N-glycosylation sites, not present in the human enzyme, are found in the primary amino acid sequence of the mouse enzyme. All seven cysteine residues in the mouse enzyme are conserved in the human enzyme. Although the nucleotide sequence could be aligned to 60% identity with the E. coli β-galactosidase, similarity in the amino acid sequence was minimal.

Effects of temperature on allosteric and catalytic properties of the cGMP-stimulated cyclic nucleotide phosphodiesterase from calf liver.

We have investigated effects of temperature on the catalytic and allosteric properties of the cGMP-stimulated cyclic nucleotide phosphodiesterase from calf liver. Vmax for cAMP and cGMP increased as assay temperature increased from 5 to 45 degrees C. At substrate concentrations below Kmapp, however, hydrolysis increased as temperature decreased from 45 to 5 degrees C and was much greater at 5 degrees C than at 45 degrees C. As assay temperature decreased, Kmapp for cAMP and cGMP decreased. Hill coefficients for cAMP and cGMP were approximately 1.9 at 45 degrees C and 1.2-1.0 at 5 degrees C. cGMP stimulated hydrolysis of 0.5 microM [3H]cAMP at all assay temperatures. Although maximal activity stimulated by cGMP, like Vmax, was lowest at 5 degrees C, presumably because of the effect of temperature on catalytic activity, the apparent activation constant (K alpha app) for cGMP stimulation was lower at 5 degrees C than at 45 degrees C. Thus, affinity for both substrate and effector was increased at 5 degrees C, suggesting that low temperature promotes transitions of the cGMP-stimulated phosphodiesterase to a "high affinity" state. That cGMP stimulated cAMP hydrolysis at 5 degrees C suggests that temperature-induced transitions are incomplete and/or readily reversible. In assays at 30 degrees C competitive inhibitors, like substrates, induce allosteric transitions which result in enhanced hydrolysis of low substrate (1.0 microM [3H] cAMP) concentrations. At higher substrate concentrations (50 microM [3H]cAMP), with the enzyme in the "activated" state, inhibitors compete with substrate at catalytic sites and reduce hydrolysis. At 45 degrees C, as at 30 degrees C, 1-methyl-3-isobutylxanthine (IBMX) and papaverine increased hydrolysis of 1.0 microM [3H]cAMP and reduced hydrolysis of 50 microM [3H]cAMP. At 5 degrees C, however, IBMX and papaverine inhibited hydrolysis of both 1.0 and 50 microM [3H]cAMP. Enzyme activity was relatively more sensitive to inhibition by IBMX at 5 degrees C than at 45 degrees C. Taken together, these observations support the notion that low temperature induces incomplete or readily reversible transitions to the high affinity state for substrates, effectors, and inhibitors. These observed effects of temperature also point out that enzyme determinants and topographical features responsible for transitions to the high affinity state and expression of catalytic activity can be regulated independently.

PH-induced hysteretic transitions of ovoperoxidase.

Ovoperoxidase, the enzyme that catalyzes the dityrosine cross-linking of fertilization membranes of eggs from the sea urchin Stronglyocentrotus purpuratus, exhibits slow changes in catalytic activity upon alterations of pH, with attendant changes in spectral properties. For ovoperoxidase pre-equilibratated at pH 8, abrupt decreases in pH are accompanied by a slow loss in activity that is temporally associated with a change in absorbance at the Soret band. With enzyme pre-equilibrated at pH 4.5 and then shifted to higher pH, there was a slow increase in catalytic activity following a rapid change in the Soret band absorbance. These changes were reversible and led to the same equilibrium state, regardless of the direction of pH shift. The rate of approach to the equilibrium state of ovoperoxidase was independent of enzyme concentration, the presence of substrates, or temperature (from 6.5 to 39.7 degrees C). The pH-induced interconversions of catalytic and spectral properties indicate that ovoperoxidase undergoes hysteretic transitions, in which alterations in the heme environment accompany, but are not sufficient for, the expression of catalytic activity. We present a kinetic mechanism for the hysteretic relaxations and suggest how these transitions may have relevance to the assembly of the fertilization membrane in vivo.

A simple rate law that describes the kinetics of the heparin-catalyzed reaction between antithrombin III and thrombin.

The kinetics of the reaction between human thrombin and antithrombin III were studied in the presence of heparin fractionated according to its molecular size and affinity for antithrombin III. Reactions were monitored continuously through the loss of fluorescence intensity accompanying the displacement of the reversible inhibitor dansylarginine-N-(3-ethyl-1,5-pentanediyl)amide from thrombin upon its interaction with antithrombin III. As the concentration of heparin was increased in the presence of fixed concentrations of the two proteins, initial rates of the reaction increased up to a maximum and then decreased. Both the maximum rate and the concentration of heparin required to obtain a maximum increased with increased concentration of the proteins. The reaction appeared second order overall and first order with respect to each protein when heparin was in excess and zero order overall when the proteins were in excess. The existence of zero order kinetics at excess concentration of proteins indicates that the reaction is saturable in both proteins. The kinetics of the heparin-catalyzed reaction measured over an appreciable range of protein and heparin concentrations could be rationalized well by the simple rate equation, v = k X fa X fb X [H]t, where k is an intrinsic rate constant for the reaction between bound antithrombin III and thrombin, fa and fb are, respectively, the fractional saturation of available sites on heparin by antithrombin III and thrombin, and [H]t is the total concentration of heparin. This rate equation implies that interactions of both proteins with heparin are involved in the expression of catalytic activity through a template mechanism as proposed by M. W. Pomeranz and W. G. Owen ((1978) Biochim. Biophys. Acta 535, 66-77) and M. J. Griffith ((1982) J. Biol. Chem. 257, 7360-7365).