Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

The contribution of (8alpha)-(N3)histidyl bond formation to the conformation of covalently flavinylated proteins was investigated by trypsin treatment of wild type and mutant versions of a model enzyme, 6-hydroxy-D-nicotine oxidase (6-HDNO) of Arthrobacter nicotinovorans. In the absence of FAD, apo-6-HDNO exhibited a conformation exposing a protease accessible site. Holoenzyme formation through FAD-attachment to His71 induced a conformational change in the protein that shielded the trypsin recognition site. This conformational change, however, did not require FAD-histidyl bond formation since trypsin resistance was also exhibited by a 6-HDNO.Cys71 mutant protein which was unable to bind FAD covalently. Replacement of Arg67, an amino acid residue supposed to be essential in flavinylation, by Ala rendered the protein protease sensitive as did replacement of Pro73 by Ala. These amino acids apparently play an essential role in stabilizing the native protein conformation. The inability to reach the native conformation also prevented FAD attachment, indicating that a specific conformation of the protein is a prerequisite for FAD-histidyl bond formation. Deletion of Phe448 and Arg449 from the 458 amino acid residues-containing enzyme resulted in complete protease sensitivity, demonstrating that flavinylation takes place posttranslationally.
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PMID:The conformational change induced by FAD in covalently flavinylated 6-hydroxy-D-nicotine oxidase does not require (8alpha)FAD-(N3)histidyl bond formation. 953 27

Protoporphyrinogen oxidase (EC 1-3-3-4), the 60-kDa membrane-bound flavoenzyme that catalyzes the final reaction of the common branch of the heme and chlorophyll biosynthesis pathways in plants, is the molecular target of diphenyl ether-type herbicides. It is highly resistant to proteases (trypsin, endoproteinase Glu-C, or carboxypeptidases A, B, and Y), because the protein is folded into an extremely compact form. Trypsin maps of the native purified and membrane-bound yeast protoporphyrinogen oxidase show that this basic enzyme (pI > 8.5) was cleaved at a single site under nondenaturing conditions, generating two peptides with relative molecular masses of 30,000 and 35,000. The endoproteinase Glu-C also cleaved the protein into two peptides with similar masses, and there was no additional cleavage site under mild denaturing conditions. N-terminal peptide sequence analysis of the proteolytic (trypsin and endoproteinase Glu-C) peptides showed that both cleavage sites were located in putative connecting loop between the N-terminal domain (25 kDa) with the betaalphabeta ADP-binding fold and the C-terminal domain (35 kDa), which possibly is involved in the binding of the isoalloxazine moiety of the FAD cofactor. The peptides remained strongly associated and fully active with the Km for protoporphyrinogen and the Ki for various inhibitors, diphenyl-ethers, or diphenyleneiodonium derivatives, identical to those measured for the native enzyme. However, the enzyme activity of the peptides was much more susceptible to thermal denaturation than that of the native protein. Only the C-terminal domain of protoporphyrinogen oxidase was labeled specifically in active site-directed photoaffinity-labeling experiments. Trypsin may have caused intramolecular transfer of the labeled group to reactive components of the N-terminal domain, resulting in nonspecific labeling. We suggest that the active site of protoporphyrinogen oxidase is in the C-terminal domain of the protein, at the interface between the C- and N-terminal domains.
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PMID:The domain structure of protoporphyrinogen oxidase, the molecular target of diphenyl ether-type herbicides. 972 41

Vitamin B2 and flavin cofactors are transported tightly bound to immunoglobulin in human serum. We reasoned that anti-mitochondrial flavoprotein autoantibodies (alpha Fp-AB) present in the serum of patients with myocarditis and cardiomyopathy of unknown aetiology may form immunoglobulin aggregates with these serum proteins. However, immunodiffusion and Western blot assays demonstrated that the flavin-carrying proteins were not recognized by alpha Fp-AB. Apparently the flavin moiety in the native protein conformation was inaccessible to alpha Fp-AB. This conclusion was supported by the absence of an immunoreaction between the riboflavin-binding protein from egg white and alpha FP-AB. Intravenous application of vitamin B2 to rabbits immunized with 6-hydroxy-D-nicotine oxidase, a bacterial protein carrying covalently attached FAD, did not neutralize alpha Fp-AB which had been raised in the serum of the animals. FAD-carrying peptides generated from 6-hydroxy-D-nicotine oxidase by trypsin and chymotrypsin treatment were not recognized by the alpha Fp-AB, but those generated by endopeptidase Lys were. This demonstrates that the epitope recognized by alpha Fp-AB comprises, besides the flavin moiety, protein secondary structure elements.
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PMID:Anti-mitochondrial flavoprotein autoantibodies of patients with myocarditis and dilated cardiomyopathy (anti-M7): interaction with flavin-carrying proteins, effect of vitamin B2 and epitope mapping. 1019 10

Monomeric sarcosine oxidase (MSOX) and N-methyltryptophan oxidase (MTOX) are homologous enzymes that catalyze the oxidative demethylation of sarcosine (N-methylglycine) and N-methyl-L-tryptophan, respectively. MSOX is induced in various bacteria upon growth on sarcosine. MTOX is an E. coli enzyme of unknown metabolic function. Both enzymes contain covalently bound flavin. The covalent flavin is at the FAD level as judged by electrospray mass spectrometry. The data provide the first evidence that MTOX is a flavoprotein. The following observations indicate that 8alpha-(S-cysteinyl)FAD is the covalent flavin in MSOX from Bacillus sp. B-0618 and MTOX. FMN-containing peptides, prepared by digestion of MSOX or MTOX with trypsin, chymotrypsin, and phosphodiesterase, exhibited absorption and fluorescence properties characteristic of an 8alpha-(S-cysteinyl)flavin and could be bound to apo-flavodoxin. The thioether link in the FMN-containing peptides was converted to the sulfone by performic acid oxidation, as judged by characteristic absorbance changes and an increase in flavin fluorescence. The sulfone underwent a predicted reductive cleavage reaction upon treatment with dithionite, releasing unmodified FMN. Cys315 was identified as the covalent FAD attachment site in MSOX from B. sp. B-0618, as judged by the sequence obtained for a flavin-containing tryptic peptide (GAVCMYT). Cys315 aligns with a conserved cysteine in MSOX from other bacteria, MTOX (Cys308) and pipecolate oxidase, a homologous mammalian enzyme known to contain covalently bound flavin. There is only one conserved cysteine found among these enzymes, suggesting that Cys308 is the covalent flavin attachment site in MTOX.
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PMID:Structure of the flavocoenzyme of two homologous amine oxidases: monomeric sarcosine oxidase and N-methyltryptophan oxidase. 1022 Mar 47

The involvement of rat liver mitochondria in the flavinylation of the mitochondrial matrix flavoenzyme dimethylglycine dehydrogenase (Me2GlyDH) has been investigated. Me2GlyDH was synthesized as an apoenzyme in the rabbit reticulocyte lysate (RL) transcription/translation system and its flavinylation was monitored by virtue of the trypsin resistance of the holoenzyme. The rate of holoenzyme formation in the presence of FAD was stimulated with increasing efficiency by the addition of solubilized mitoplasts, mitochondrial matrix and DEAE-purified matrix fraction. Apo-Me2GlyDH was also converted into holoenzyme when the solubilized mitoplasts were supplemented with FMN and ATP. This observation is consistent with the existence of a mitochondrial FAD synthetase generating the FAD needed for holoenzyme formation from its precursors. Holoenzyme formation in the presence of FAD increased linearly with the concentration of matrix protein in the assay, and depended on the amount of externally added Me2GlyDH with saturation characteristics. These findings suggest the presence of a protein factor in the mitochondrial matrix which stimulates Me2GlyDH flavinylation. This factor was different from both mitochondrial heat shock protein (Hsp)70, as shown by immunodepletion experiments, and mitochondrial Hsp60, as demonstrated by the capability of a DEAE-purified matrix fraction devoid of Hsp60 to accelerate flavinylation of both RL translated and purified Me2GlyDH.
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PMID:A protein factor of rat liver mitochondrial matrix involved in flavinylation of dimethylglycine dehydrogenase. 1088 Sep 57

Monoamine oxidase is a flavin-containing enzyme located at the mitochondrial outer membrane that catalyzes the oxidative deamination of amines. To investigate the role of tyrosine residues near the FAD-binding site, Cys-406, of monoamine oxidase A, the tyrosine residues at posiyions 402, 407, and 410 were indurdually replaced with alanine or phenylalanine and the effects of the mutations on catalytic activity, FAD binding, and enzyme structure were examined. Half or fewer of the mutant proteins incorporated FAD. The mutation of Tyr-407 to alanine led to an almost completely loss of catalytic activity for serotonin, PEA, tyramine, and tryptamine. A substantial decrease in the catalytic activity was also observed with the enzymes mutated at Tyr-402 and Tyr-410 to alanine, although the effect of the latter mutation was much less. All these mutants were sensitive to trypsin treatment of the purified enzyme, while the wild type enzyme was resistant to treatment. On the other hand, substitution of Tyr-402 or Tyr-407 with phenylalanine had little effect on these properties. Taken together, we conclude that tyrosine residues near Cys-406 may be form a pocket to facilitates FAD incorporation, the catalytic center, and a stable conformation, probably through interactions among the aromatic rings of the tyrosine residues and FAD.
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PMID:Tyrosine residues near the FAD binding site are critical for FAD binding and for the maintenance of the stable and active conformation of rat monoamine oxidase A. 1175 41

Plasmids were constructed for overexpression of the Escherichia coli dihydrolipoamide acetyltransferase (1-lip E2, with a single hybrid lipoyl domain per subunit) and dihydrolipoamide dehydrogenase (E3). A purification protocol is presented that yields homogeneous recombinant 1-lip E2 and E3 proteins. The hybrid lipoyl domain was also expressed independently. Masses of 45,953+/-73Da (1-lip E2), 50,528+/-5.5Da (apo-E3), 51,266+/-48Da (E3 including FAD), and 8982+/-4.0 (lipoyl domain) were determined by MALDI-TOF mass spectrometry. The purified 1-lip E2 and E3 proteins were functionally active according to the overall PDHc activity measurement. The lipoyl domain was fully acetylated after just 30 s of incubation with E1 and pyruvate. The mass of the acetylated lipoyl domain is 9019+/-2Da according to MALDI-TOF mass spectrometry. Treatment of the 1-lip E2 subunit with trypsin resulted in the appearance of the lipoyl domain with a mass of 10,112+/-3Da. When preincubated with E1 and pyruvate, this tryptic fragment was acetylated according to the mass increase. MALDI-TOF mass spectrometry was thus demonstrated to be a fast and precise method for studying the reductive acetylation of the recombinant 1-lip E2 subunit by E1 and pyruvate.
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PMID:Expression and purification of the dihydrolipoamide acetyltransferase and dihydrolipoamide dehydrogenase subunits of the Escherichia coli pyruvate dehydrogenase multienzyme complex: a mass spectrometric assay for reductive acetylation of dihydrolipoamide acetyltransferase. 1265 Nov 18

The flavoenzyme d-amino acid oxidase (DAAO) from Rhodotorula gracilis is a peroxisomal enzyme and a prototypical member of the glutathione reductase family of flavoproteins. DAAO is a stable homodimer with a FAD molecule tightly bound to each 40-kDa subunit. In this work, the urea-induced unfolding of dimeric DAAO was compared with that of a monomeric form of the same protein, a deleted dimerization loop mutant. By using circular dichroism spectroscopy, protein and flavin fluorescence, 1,8-anilinonaphtalene sulfonic acid binding and activity assays, we demonstrated that the urea-induced unfolding of DAAO is a three-state process, yielding an intermediate, and that this process is reversible. The intermediate species lacks the catalytic activity and the characteristic tertiary structure of native DAAO but has significant secondary structure and retains flavin binding. Unfolding of DAAO proceeds through formation of an expanded, partially unfolded inactive intermediate, characterized by low solubility, by increased exposure of hydrophobic surfaces, and by increased sensitivity to trypsin of the beta-strand F5 belonging to the FAD binding domain. The oligomeric state does not modify the inferred folding process. The strand F5 is in contact with the C-terminal alpha-helix containing the Ser-Lys-Leu sequence corresponding to the type 1 peroxisomal targeting signal, and this structural element interacts with the N-terminal betaalphabeta flavin binding motif (Rossmann fold). The expanded conformation of the folding intermediate (and in particular the higher disorder of the mentioned secondary structure elements) could match the structure of the inactive holoenzyme required for in vivo trafficking of DAAO through the peroxisomal membrane.
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PMID:Unfolding intermediate in the peroxisomal flavoprotein D-amino acid oxidase. 1510 41

The nucleotide sequence encoding L-phenylalanine oxidase (deaminating and decarboxylating) (PAO) from Pseudomonas sp. P-501 was determined. The open reading frame is arranged in the order of prosequence, alpha subunit, dipeptide and beta subunit from the 5'- to 3'-end. Expression of the gene in Escherichia coli showed that without the prosequence, PAO is produced in small quantity as a soluble form with no visible absorption, but with the prosequence (proPAO), PAO is highly expressed and yellow. The purified proPAO contained one mol of FAD per mol of proPAO polypeptide, but had no catalytic activity. Treatment of proPAO with a mixture of Pronase and trypsin converted the noncatalytic proPAO to the catalytic form, and the Pronase-trypsin-treated proPAO showed kinetic and spectral properties comparable to the native enzyme. These results suggest that in Pseudomonas, PAO is expressed as a proenzyme that is processed by proteolysis to the active form.
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PMID:Sequencing and expression of the L-phenylalanine oxidase gene from Pseudomonas sp. P-501. Proteolytic activation of the proenzyme. 1563 1

Hexose oxidase (EC 1.1.3.5) from Hansenula polymorpha was found to exhibit a dual covalent association of FAD with His79 via an 8 alpha-histidyl linkage as well as a covalent association between Cys138 and C-6 of the isoalloxazine moiety of FAD. Spectral properties of the wild-type enzyme exhibited maxima at 364 nm and 437 nm as well as a distinct shoulder at 445 nm. An H79K mutant enzyme exhibited only one maximum at 437 nm. The difference absorption spectrum between an oxidized and a substrate-reduced enzyme preparation showed maxima at 360 nm and 445 nm corresponding to an apparent novel type of association. Hexose oxidase showed a low, pH-independent fluorescence at 525 nm when excited at 450 nm. Flavin was released from the holoenzyme by treatment with trypsin. Sequencing of the flavopeptide revealed two peptides comprising positions 74-91 and 132-157 associated with FAD in equimolar amounts. A homology model of hexose oxidase was constructed using the crystal structure of glucooligosaccharide oxidase from Acremonium strictum as template. The model placed both of the sequences found above in the close vicinity of the FAD cofactor, and suggests covalent bonds between both His79 and Cys138 and FAD, in accordance with the chemical evidence. Based on the results, hexose oxidase is identified as incorporating FAD with a double covalent association with His79 and Cys138 in the holoenzyme. A reaction mechanism involving the concerted action of Tyr488 and Asp409 in hexose oxidase is suggested as the initiator of the proton abstraction from the substrate molecule in the active site.
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PMID:Characterization of the flavin association in hexose oxidase from Chondrus crispus. 1681 97


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