Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.24.27 (thermolysin)
 1,894 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase catalyzes exchange reactions between ADP and ATP and between fructose-6-P and fructose-2,6-P2 at histidyl residues. Limited proteolysis of the enzyme with thermolysin yielded an enzyme core with a subunit molecular weight of 35,000-38,000. This enzyme core had no kinase activity and a 2-fold activated bisphosphatase activity whose sensitivity to the product inhibitor fructose-6-P was unchanged. The thermolysin-treated enzyme also did not catalyze the fructose-6-P/fructose-2,6-P2 exchange reaction but did catalyze the ADP/ATP exchange. These results suggest that 1) the enzyme's reactions may be catalyzed at two active sites, 2) there are at least two fructose-6-P binding sites, 3) the fructose-6-P/fructose-2,6-P2 exchange is catalyzed only at the kinase site, and 4) inactivation of the exchange and kinase reactions by thermolysin digestion is due to the loss of the fructose-6-P binding site of the kinase. Also consistent with these conclusions was the finding that oxidation of the enzyme with ascorbate/Fe3+ or H2O2 resulted in complete loss of the kinase activity as well as the fructose-6-P/fructose-2,6-P2 exchange but did not affect the bisphosphatase activity or the ADP/ATP exchange. Dithiothreitol could completely reactivate the ascorbate/Fe3+-inactivated enzyme, suggesting that oxidation occurred at a sulfhydryl group(s) essential for fructose-6-P binding in the kinase reaction. In addition, the kinase and fructose-6-P/fructose-2,6-P2 exchange reactions were more sensitive to inactivation by diethylpyrocarbonate than was the bisphosphatase. The different responses of the kinase and bisphosphatase reactions to the action of these various protein-modifying agents and to thermolysin digestion support the existence of a separate site for each reaction and an essential role for sulfhydryl groups at the sugar-phosphate-binding site(s) of the kinase.
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PMID:Differential effects of proteolysis and protein modification on the activities of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. 609 63

Both the synthesis and the degradation of Fru-2,6-P2 are catalyzed by a single enzyme protein; ie, the enzyme is bifunctional. This protein, which we have designated 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase is an important enzyme in the regulation of hepatic carbohydrate metabolism since its activity determines the steady-state concentration of fructose 2,6-P2, an activator of 6-phosphofructo 1-kinase and an inhibitor of fructose 1,6-bisphosphatase. Regulation of the bifunctional enzyme in intact cells is a complex function of both covalent modification via phosphorylation/dephosphorylation and the influence of substrates and low molecular weight effectors. Recent evidence suggests that both reactions may proceed by two-step transfer mechanisms with different phosphoenzyme intermediates. The enzyme catalyzes exchange reactions between ADP and ATP and between fructose 6-P and fructose 2,6-P2. A labeled phosphoenzyme is formed rapidly during incubation with [2-32P]Fru-2,6-P2. The labeled residue has been identified as 3-phosphohistidine. However, it was not possible to demonstrate significant labeling of the enzyme directly from [gamma-32P]ATP. These results can be most readily explained in terms of two catalytic sites, a kinase site whose phosphorylation by ATP is negligible (or whose E-P is labile) and a fructose 2,6-bisphosphatase site which is readily phosphorylated by fructose 2,6-P2. Additional evidence in support of two active sites include: limited proteolysis with thermolysin results in loss of 6-phosphofructo 2-kinase activity and activation of fructose 2,6-bisphosphatase, mixed function oxidation results in inactivation of the 6-phosphofructo 2-kinase but no affect on the fructose 2,6-bisphosphatase, N-ethylmaleimide treatment also inactivates the kinase but does not affect the bisphosphatase, and p-chloromercuribenzoate immediately inactivates the fructose 2,6-bisphosphatase but not the 6-phosphofructo 2-kinase. Our findings indicate that the bifunctional enzyme is a rather complicated enzyme; a dimer, probably with two catalytic sites reacting with sugar phosphate, and with an unknown number of regulatory sites for most of its substrates and products. Three enzymes from Escherichia coli, isocitric dehydrogenase kinase/phosphatase, glutamine-synthetase adenylyltransferase, and the uridylyltransferase for the regulatory protein PII in the glutamine synthetase cascade system also catalyze opposing reactions probably at two discrete sites. All four enzymes are important in the regulation of metabolism and may represent a distinct class of regulatory enzymes.
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PMID:Rat liver 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase: a review of relationships between the two activities of the enzyme. 609 84

Pseudomonas toxin is produced as a proenzyme which is cytotoxic for cells in culture but must be activated to express full enzymatic activity. The ability of purified pseudomonas alkaline protease and elastase or of culture filtrates of two strains of Pseudomonas aeruginosa to modify the activity of pseudomonas toxin was examined. Two parameters of toxin activity were followed: enzymatic activity, i.e., the adenosine diphosphate (ADP) ribosylation of elongation factor 2, and biological activity, i.e., inhibition of protein synthesis in cultured mouse fibroblasts. Biological activity of toxin depends upon an intact toxin molecule, whereas enzyme activity requires only a functional A region. Incubation with purified pseudomonas proteolytic enzymes did not alter either enzymatic or biological activity. The toxin is not refractory to the action of all proteolytic enzymes, since thermolysin rapidly destroyed the toxin molecule. Treatment of toxin with culture filtrates of P. aeruginosa reduced ADP ribosylation activity, but increased the ability of toxin to inhibit protein synthesis in cell monolayers. Incubation of culture filtrates with one of the protease inhibitors alpha-2-macroglobulin or phosphoramidon did not alter the effect of the filtrates on biological activity. Alpha-2-macroglobulin, however, caused a fourfold stimulation of ADP ribosylation activity of the toxin. We conclude that pseudomonas alkaline protease and elastase are not responsible for the modifications in toxin activity induced by culture filtrates of P. aeruginosa; the factors responsible have not yet been identified, but are not inactivated by phosphoramidon or alpha-2-macroglobulin.
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PMID:Resistance of exotoxin A to purified Pseudomonas proteolytic enzymes. 615 7

The effects of various proteases on the enzymatic or biological activity and structure of exotoxin A from Pseudomonas aeruginosa were systematically studied. The toxin was extremely resistant to treatment with various enzymes. The lethality of the toxin disappeared upon treatment with P. aeruginosa protease and elastase, thermolysin, and trypsin with a long incubation time (5h) in the presence of a high enzyme concentration (molar concentration of enzyme to toxin, 1:10 or 1:20), but was little altered by either alpha-chymotrypsin or subtilisin. The decrease of adenosine diphosphate ribosylation activity was moderate when the same treatment was used, regardless to the protease source, except in the case of papain, which was tested in the presence of reducing agents. The increase in activation of the treated toxin determined in the presence of a denaturant and a reducing agent was less than that of the intact toxin, except in the case of trypsin. The differences in disc and sodium dodecyl sulfate gel electropherograms of the toxins treated with these proteases, except for those treated with papain, suggested that the toxins had been nicked by the protease, which resulted in their degradation by sodium dodecyl sulfate treatment. Papain degraded the toxin into fragments and caused the disappearance of lethality or a marked decrease of adenosine diphosphate ribosylation activity.
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PMID:Effects of proteases on the structure and activity of Pseudomonas aeruginosa exotoxin A. 703 Sep 58

The atomic structure of myosin subfragment-1 (S1) has been recently solved for crystals of extensively methylated S1 [Rayment et al. (1993) Science 261, 50-58]. In this study, the effect of such a modification on S1 structure and function was examined. According to the far- and near-ultraviolet CD spectra, the methylation does not affect the secondary structure of S1 but causes limited changes in its tertiary structure. The methylation significantly decreases the affinity of S1 for actin in rigor and, to a lesser degree, that of S1 to actin in the presence of MgATP gamma S. This modification, like the trinitrophenylation of Lys-83, accelerates the dissociation of a nucleotide trapped on S1 either by phosphate analogs or by cross-linking of the SH1 and SH2 thiols. Methylation strongly impairs the coupling between the actin- and nucleotide-binding sites as revealed by the reduced effect of actin on the release of epsilon ADP from the active site. It also causes a complete loss of in vitro motility of actin filaments over methylated HMM. In addition to this, methylation also impairs the communication between other sites on S1 including that between the nucleotide-binding site and SH1, and the actin-binding site and the 27/50 kDa junction and a site at 74 kDa from the N-terminus of S1. These changes are revealed in SH1 modification, thermolysin digestion, and vanadate-dependent photocleavage experiments, respectively. The increased rate of thermal denaturation of S1 and the loss of S1 protection by ADP and actin from this process also indicate flawed communications in methylated S1. It is concluded that these relatively mild but numerous and important changes impair the function of methylated S1.
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PMID:Extensively methylated myosin subfragment-1: examination of local structure, interactions with nucleotides and actin, and ligand-induced conformational changes. 772 79

A central question in molecular biology concerns the means by which a regulatory protein recognizes different targets. IIIGlc, the glucose-specific phosphocarrier protein of the bacterial phosphotransferase system, is also the central regulatory element of the PTS. Binding of unphosphorylated IIIGlc inhibits several non-PTS proteins, but there is little or no sequence similarity between IIIGlc binding sites on different target proteins. The crystal structure of Escherichia coli IIIGlc bound to one of its regulatory targets, glycerol kinase, has been refined at 2.6-A resolution in the presence of products, adenosine diphosphate and glycerol 3-phosphate. Structural and kinetic analyses show that the complex of IIIGlc with glycerol kinase creates an intermolecular Zn(II) binding site with ligation identical to that of the zinc peptidase thermolysin. The zinc is coordinated by the two active-site histidines of IIIGlc, a glutamate of glycerol kinase, and a water molecule. Zn(II) at 0.01 and 0.1 mM decreases the Ki of IIIGlc for glycerol kinase by factors of about 15 and 60, respectively. The phosphorylation of one of the histidines of IIIGlc, in its alternative role as phosphocarrier, provides an elegant means of controlling the cation-enhanced protein-protein regulatory interaction. The need for the target protein to supply only one metal ligand may account for the lack of sequence similarity among the regulatory targets of IIIGlc.
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PMID:Cation-promoted association of a regulatory and target protein is controlled by protein phosphorylation. 817 Sep 44

We used a battery of proteases to probe the footprint of microtubules on kinesin and ncd, and to search for nucleotide-induced conformational changes in these two oppositely-directed yet homologous molecular motors. Proteolytic cleavage sites were identified by N-terminal microsequencing and electrospray mass spectrometry, and then mapped onto the recently-determined atomic structures of ncd and kinesin. In both kinesin and ncd, microtubule binding shields a set of cleavage sites within or immediately flanking the loops L12, L8 and L11 and, in ncd, the loop L2. Even in the absence of microtubules, exchange of ADP for AMPPNP in the motor active site drives conformational shifts involving these loops. In ncd, a chymotryptic cleavage at Y622 in L12 is protected in the strong binding AMPPNP conformation, but cleaved in the weak binding ADP conformation. In kinesin, a thermolysin cleavage at L154 in L8 is protected in AMPPNP but cleaved in ADP. We speculate that ATP turnover in the active site governs microtubule binding by cyclically retracting or displaying the loops L8 and L12. Curiously, the retracted state of the loops corresponds to microtubule strong binding. Conceivably, nucleotide-dependent display of loops works as a reversible block on strong binding.
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PMID:Proteolytic mapping of kinesin/ncd-microtubule interface: nucleotide-dependent conformational changes in the loops L8 and L12. 946 73

Renin-binding protein (RnBP) is an endogenous renin inhibitor originally isolated from porcine kidney as a complex of renin, so-called high molecular weight (HMW) renin. Our recent studies demonstrated that human RnBP is the enzyme N-acetyl-D-glucosamine (GlcNAc) 2-epimerase [Takahashi, S. et al. (1999) J. Biochem. 125, 348-353]. We have purified recombinant human, rat, and porcine RnBPs expressed in Escherichia coli JM 109 cells. The purified recombinant RnBPs existed as dimers and inhibited porcine renin activity strongly. On the other hand, porcine renin inhibited recombinant GlcNAc 2-epimerase activities. The human GlcNAc 2-epimerase activity could not be detected in the absence of a nucleotide, whereas ATP, dATP, ddATP, ADP, and GTP enhanced the human GlcNAc 2-epimerase activity. Other nucleotides had no effect on human GlcNAc 2-epimerase activity. Rat and porcine GlcNAc 2-epimerases were activated by several nucleotides. Nucleotides that enhance the activity of GlcNAc 2-epimerases protect these enzymes against degradation by thermolysin. These results indicate that mammalian RnBPs have GlcNAc 2-epimerase activity and that nucleotides are essential for formation of the catalytic domain of the enzyme.
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PMID:Effects of nucleotides on N-acetyl-d-glucosamine 2-epimerases (renin-binding proteins): comparative biochemical studies. 1172 82


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