Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.4 (trypsin)
 42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Phosphoenolpyruvate carboxylase from Escherichia coli W was treated with ten proteases, and the effects of the treatments on the enzyme activity and sensitivity to effectors were investigated. Proteases such as trypsin, alpha-chymotrypsin, papain, and subtilisin inactivated the enzyme, whereas elastase, carboxypeptidase Y and leucine aminopeptidase had no effect on the enzyme activity. Elastase and carboxypeptidase Y, however, inactivated the enzyme in the presence of 1 m urea. Subtilisin and alpha-chymotrypsin caused not only inactivation of the enzyme but also a significant desensitization to the effectors. DL-Phospholactate, a potent competitive inhibitor, markedly protected the enzyme from inactivation by subtilisin but did not protect it from desensitization to the effectors. Acetyl-CoA, fructose 1, 6-bisphosphate, and GTP-the allosteric activators--protected the enzyme from subtilisin inactivation, while laurate, the other allosteric activator, accelerated the inactivation. These activators did not protect the enzyme from desensitization to themselves. In contrast, modification with subtilisin in the present of l-aspartate, the allosteric inhibitor, caused an apparent transient activation of the enzyme. The enzyme modified in the presence of L-aspartate retained its sensitivity to L-aspartate, but the sensitivities to the other effectors were reduced to about one-half their initial values. Based on these results, a possible mode of desensitization of the enzyme by subtilisin modification and the possible existence of a multiplicity of conformational states of the enzyme, induced upon binding with the various effectors, are discussed.
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PMID:Phosphoenolpyruvate carboxylase of Escherichia coli. Effect of proteolytic modification on the catalytic and regulatory propties. 38 5

1. Limited proteolysis of citrate synthase from Sulfolobus solfataricus by trypsin reduced the rate of the overall reaction (acetyl-CoA + oxaloacetate + H2O----citrate + CoASH) to 4% but did not affect the hydrolysis of citryl-CoA. Experimental results indicate that a connecting link between the enzyme's ligase and hydrolase activity becomes impaired specifically on treatment with trypsin. Other proteolytic enzymes like chymotrypsin and subtilisin inactivated catalytic functions of citrate synthase, ligase and hydrolase, equally well. 2. Tryptic hydrolysis occurs at the N-terminal region of citrate synthase, but a study by SDS/PAGE revealed no difference in molecular mass between native and proteolytically nicked citrate synthase. The peptide removed from the enzyme by trypsin, therefore, contains less than about 15 amino acid residues. 3. The Km values of the substrates for both native and nicked enzyme were identical, as was the state of aggregation (dimeric) of the two enzyme species. These could be separated by affinity chromatography on Blue-Sepharose and differentiated by their isoelectric points (pI = 6.68 +/- 0.08 and pI = 6.37 +/- 0.03 for native citrate synthase and the large tryptic peptide, respectively) as well as by the N-terminus which is blocked in the native enzyme only. 4. Edman degradation of the large tryptic fragment yielded the N-terminal sequence GLEDVYIKSTSLTYIDGVNGVLRY, which is 71% identical to the N-terminal region (positions 9-32) of citrate synthase from Thermoplasma acidophilum. 5. The conversion of citrate synthase into essentially a citryl-CoA hydrolase is considered the consequence of a conformational change thought to occur on tryptic removal of the N-terminal small peptide.
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PMID:Conversion, by limited proteolysis, of an archaebacterial citrate synthase into essentially a citryl-CoA hydrolase. 152 37

Native rat liver methylmalonate semialdehyde dehydrogenase was proteolyzed by lysylendopeptidase C, chymotrypsin, and trypsin to generate different cleavage fragments of molecular masses: 50, 8, 55, 44, 39, 53, 45, and 40 kDa. A proteolytic cleavage map of MMSDH was constructed based on sequencing data and a comparison of appearance and degradation rates of the different protein fragments as shown by SDS-PAGE. NAD+ was highly effective as a protector against proteolysis in both the N-terminal and the C-terminal parts of the intact enzyme. NADH did not efficiently protect the intact enzyme; however, it stabilized proteolytic fragment L50 from further degradation. This suggests that the NAD(+)-binding domain is not destroyed by cleavage of the N-terminal part of MMSDH. CoA had no effect on the proteolytic cleavage patterns of MMSDH. However, CoA esters reduced the protective effect of NAD+ with an order of effectiveness of acetyl-CoA greater than propionyl-CoA greater than butyryl-CoA. p-Nitrophenyl acetate, substrate for esterase activity by the enzyme, partially prevented the protective effect of NAD+ against proteolysis. These results suggest that S-acylation of the enzyme prevents a stabilizing conformational change induced in MMSDH by NAD+ binding.
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PMID:The effect of ligand binding on the proteolytic pattern of methylmalonate semialdehyde dehydrogenase. 189 92

Pyruvate:NADP+ oxidoreductase from Euglena gracilis, a homodimeric protein with a molecular weight of 309 kDa, is an iron-sulfur flavoenzyme that contains thiamin pyrophosphate (TPP). The functional structure of the enzyme was studied by a limited proteolysis experiment using trypsin. The evidence obtained shows that the enzyme consists of two functional domains, one of which contains an iron-sulfur cluster, which can be isolated as a homodimeric fragment of approximately 220 kDa by proteolysis. The other domain that contains FAD is released as a monomeric fragment of approximately 55 kDa. The pyruvate dehydrogenase reaction is still catalyzed by the large fragment when NADP+ is substituted by methyl viologen, while the small fragment retains a diaphorase-like electron-transfer activity from NADPH to MV. It is thus shown that pyruvate is oxidized in a CoA-dependent reaction to form CO2 and acetyl-CoA in the iron-sulfur domain, and that the two electrons formed are transferred to the FAD domain in which NADP+ is reduced. TPP is considered to be associated in the iron-sulfur domain. The NH2-terminal sequences of the enzyme and its proteolytic fragments reveal that the iron-sulfur domain occurs in the NH2-terminal side of the enzyme. For elucidation of the O2 instability of the enzyme, limited proteolysis was attempted in air. The tryptic fragment derived from the iron-sulfur domain, similar to the native enzyme, appears to be inactivated by direct contact with O2. In contrast, the FAD domain, when separated from the other domain, is quite stable in air, although the diaphorase activity decays when the native enzyme is exposed to O2.
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PMID:Pyruvate:NADP+ oxidoreductase from Euglena gracilis: limited proteolysis of the enzyme with trypsin. 191 Feb 87

Pure 2-amino-3-ketobutyrate CoA ligase from Escherichia coli, which catalyzes the cleavage/condensation reaction between 2-amino-3-ketobutyrate (the presumed product of the L-threonine dehydrogenase-catalyzed reaction) and glycine + acetyl-CoA, is a dimeric enzyme (Mr = 84,000) that requires pyridoxal 5'-phosphate as coenzyme for catalytic activity. Reduction of the hololigase with tritiated NaBH4 yields an inactive, radioactive enzyme adduct; acid hydrolysis of this adduct allowed for the isolation and identification of epsilon-N-pyridoxyllysine. Quantitative determinations established that 2 mol of pyridoxal 5'-phosphate are bound per mol of dimeric enzyme. After the inactive, tritiated enzyme adduct was digested with trypsin, a single radioactive peptide containing 23 amino acids was isolated and found to have the following primary structure: Val-Asp-Ile-Ile-Thr-Gly-Thr-Leu-Gly-Lys*-Ala-Leu-Gly-Gly-Ala-Ser-Gly-Gly -Tyr-Thr-Ala-Ala-Arg (where * = the lysine residue in azomethine linkage with pyridoxal 5'-phosphate). This peptide corresponds to residues 235-257 in the intact protein; 10 residues around the lysine residue have a high level of homology with a segment of the primary structure of 5-aminolevulinate synthase from chicken liver.
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PMID:2-Amino-3-ketobutyrate CoA ligase of Escherichia coli: stoichiometry of pyridoxal phosphate binding and location of the pyridoxyllysine peptide in the primary structure of the enzyme. 210 56

ATP:citrate lyase was purified from the oleaginous yeast Rhodotorula gracilis to homogeneity as judged by polyacrylamide gel electrophoresis, using a novel citrate-Sepharose procedure. The enzyme was found to have a molecular weight of 520,000 and consisted of four identical subunits (Mr = 120,000). Two minor low molecular weight bands were observed on SDS-PAGE (Mr 51,000 and 49,000). Trypsin digestion experiments indicated that these could have been the result of limited proteolysis by an endogenous trypsin-like proteinase. In this respect, it resembles the mammalian ATP:citrate lyase. The enzyme was stimulated by NH+4 ions and inhibited by palmitoyl, lauroyl, oleoyl, myristoyl and stearoyl-CoA esters, glutamate and glucose 6-phosphate but not by acetyl-CoA or shorter chain fatty acyl-CoA esters. The enzyme exhibited normal Michaelis-Menten kinetics for citrate; however there was a 3-fold increase in Km with a high concentration of Cl- ions (0.25 M). The possible regulatory roles of ATP:citrate lyase in R. gracilis are discussed in the light of these findings.
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PMID:ATP:citrate lyase of Rhodotorula gracilis: purification and properties. 230 11

Cell membrane contact induces the de novo expression of choline O-acetyltransferase (CAT; acetyl-CoA: choline O-acetyltransferase, EC 2.3.1.6) activity in cultures of virtually pure neonatal rat dissociated sympathetic neurons. To identify molecular mechanisms underlying membrane-associated CAT induction, the responsible membrane component was characterized and partially purified. Substantial CAT-inducing activity was found in membranes from adult rat spinal cord and sensory and sympathetic ganglia. Whole brain membranes demonstrated significantly less activity. CAT induction in sympathetic neurons in response to spinal cord membranes was linear with respect to time, after an initial 6-hr lag. It was also linear with respect to concentrations of spinal cord protein from 2 to 100 micrograms per ml. CAT-inducing activity was extracted from spinal cord membranes by incubation with 100 mM NaCl and was purified approximately 5000-fold by DEAE ion-exchange and gel filtration chromatography. The active factor appears to be an extrinsic protein with an apparent molecular mass of 27 kDa. It is inactivated by trypsin and chymotrypsin but is moderately thermostable, retaining activity at 60 degrees C but not at 90 degrees C.
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PMID:Partial purification and characterization of a membrane-derived factor regulating neurotransmitter phenotypic expression. 256 90

Choline acetyltransferase (Acetyl-CoA:choline O-acetyltransferase, EC 2.3.1.6, abbreviated ChAT), the biosynthetic enzyme for acetylcholine and acetylcholinesterase (EC 3.1.1.7, abbreviated AChE) are expressed in a human cholinergic neuroblastoma cell line, MC-IXC. We have shown that ChAT activity can be regulated in culture by retinoic acid, an active metabolite of vitamin A, and by sodium butyrate, an organic fatty acid. Optimal concentrations of these agents produce 4.3-fold and 1.6-fold increases in ChAT activity, respectively. The effects of retinoic acid are statistically significant after 24 h, whereas for sodium butyrate significant differences are seen only after 48 h. Since retinoic acid stimulation of ChAT activity was reversed only by trypsin treatment and not by removal of retinoic acid from the medium, this suggests that this agent may be acting at the level of the cell surface. Other differentiating conditions, such as culture in serum-free medium or addition of 1-2% dimethylsulfoxide did not increase ChAT activity. Acetylcholinesterase activity was shown to increase only in the presence of sodium butyrate, suggesting that retinoic acid and sodium butyrate may be acting via different pathways. Retinoic acid and sodium butyrate both seem to be permissive rather than instructive in regulating ChAT activity in that they are unable to induce ChAT expression de novo in cell lines which do not already express ChAT activity.
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PMID:Stimulation of choline acetyltransferase activity by retinoic acid and sodium butyrate in a cultured human neuroblastoma. 292 23

Fatty acid synthase from the uropygial gland was inactivated by treatment with pyrenebutyl methanephosphonofluoridate by specific modification of the "active serine" at the thioesterase domain. Treatment of fatty acid synthase with 3-(4-maleimidylphenyl)-7-diethylamino-4-methylcoumarin resulted in the loss of the condensation activity and overall synthase activity. Acetyl-CoA and malenyl-CoA protected the enzyme from inactivation by this reagent suggesting that the pantetheine thiol was modified. In support of this conclusion was the finding that modification of the primer-binding thiol with iodoacetamide prior to the modification with the coumarin derivative resulted in no change in the binding of the coumarin to the enzyme. Furthermore, the presumptive active site peptide isolated after proteolysis released its attached coumarin upon treatment with alkali under beta-elimination reaction conditions. Graphical analysis of the binding data suggested that binding of one coumarin derivative/subunit of the synthase would result in complete loss of the synthase activity. When the synthase was modified with the coumarin and pyrene derivatives, fluorescence resonance energy transfer occurred from the pyrene at the thioesterase site to the coumarin attached to the pantetheine thiol. Dissociation of the enzyme to monomers did not decrease the efficiency of transfer, but limited trypsin treatment, which released the thioesterase domain, abolished the fluorescence resonance energy transfer. These results suggested that the energy transfer occurred between intrasubunit sites. The distance between the pyrene at the thioesterase active site and the coumarin attached to pantetheine thiol on the same subunit of fatty acid synthase was estimated from the efficiency of energy transfer to be 37 A.
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PMID:Measurement of distance between the active serine of the thioesterase domain and the pantetheine thiol of fatty acid synthase by fluorescence resonance energy transfer. 391 10

The mechanism of acyl enzyme formation from acyl-CoA derivatives was studied for chicken liver fatty acid synthase in 0.1 M potassium phosphate (pH 7.0) and 1 mM EDTA at 23 degrees C. Three mechanistically important acyl-binding sites exist: a cysteine, 4'-phosphopantetheine, and a hydroxyl (serine). The cysteine was specifically labeled with iodoacetamide, and chemical modification of this labeled enzyme with chloroacetyl-CoA resulted in additional covalent labeling of 4'-phosphopantetheine. Reaction of the enzyme with acetyl-CoA results in 47% oxyester formation, whereas with malonyl-CoA and butyryl-CoA, 57 and 80% are oxyesters, respectively, as judged by treatment of the denatured enzyme with hydroxylamine. Limited proteolysis with trypsin followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that the reactive hydroxyl and cysteine are on the same peptide. Butyryl-CoA is a relatively poor primer for steady state fatty acid synthesis, probably because transfer from the hydroxyl-binding site to 4'-phosphopantetheine is inefficient. Quenched flow studies indicate that the rate constants for transfer of acetyl from enzyme-bound acetyl-CoA to native, iodoacetamide-labeled, and iodoacetamide-chloroacetyl-labeled enzyme are 43, 110, and 150 s-1. These results can be interpreted in terms of a random acylation of the hydroxyl, 4'-phosphopantetheine, and cysteine by enzyme-bound acetyl-CoA with rate constants of 150 s-1, less than 110 s-1, and less than 43 s-1, respectively. Alternatively the latter two rate constants could be characteristic of intramolecular transfer between enzyme acylation sites. Structural constraints apparently prevent all three acylation sites from being occupied simultaneously. The rate of deacetylation of the acetylated enzyme by enzyme-bound CoA also is most rapid for the iodoacetamide-chloroacetyl-labeled enzyme.
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PMID:Elementary steps in the reaction mechanism of chicken liver fatty acid synthase. Acylation of specific binding sites. 405 47


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