Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.1 (chymotrypsin)
 10,938 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cholesterol oxidase [EC 1.1.3.6] from Schizophyllum commune was purified by an affinity chromatography using 3-O-succinylcholesterol-ethylenediamine (3-cholesteryl-3-[2-aminoethylamido]propionate) Sepharose gels. The resulting preparation was homogeneous as judged by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 53,000 by SDS-gel electrophoresis and 46,000 by sedimentation equilibrium. The enzyme contained 483 amino acid residues as calculated on the basis of the molecular weight of 53,000. The enzyme consumed 60 mumol of O2/min per mg of protein with 1.3 mM cholesterol at 37 degrees C. The enzyme showed the highest activity with cholesterol; 3 beta-hydroxysteroids, such as dehydroepiandrosterone, pregnenolone, and lanosterol, were also oxidized at slower rates. Ergosterol was not oxidized by the enzyme. The Km for cholesterol was 0.33 mM and the optimal pH was 5.0. The enzyme is a flavoprotein which shows a visible absorption spectrum having peaks at 353 nm and 455 nm in 0.1 M acetate buffer, pH 4.0. The spectrum was characterized by the hypsochromic shift of the second absorption peak of the bound flavin. The bound flavin was reduced on anaerobic addition of a model substrate, dehydroepiandrosterone. Neither acid not heat treatment released the flavin coenzyme from the enzyme protein. The flavin of the enzyme could be easily released from the enzyme protein in acid-soluble form as flavin peptides when the enzyme protein was digested with trypsin plus chymotrypsin. The mobilities of the aminoacyl flavin after hydrolysis of the flavin peptides on thin layer chromatography and high voltage electrophoresis differed from those of free FAD, FMN, and riboflavin. A pKa value of 5.1 was obtained from pH-dependent fluorescence quenching process of the aminoacyl flavin. AMP was detected by hydrolysis of the flavin peptides with nucleotide pyrophosphatase. The results indicate strongly that cholesterol oxidase from Schizophyllum commune contains FAD as the prothetic group, which is covalently linked to the enzyme protein. The properties of the bound FAD were comparable to those of N (1)-histidyl FAD.
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PMID:Purification and some properties of cholesterol oxidase from Schizophyllum commune with covalently bound flavin. 3 75

Brain pyridoxine-5-P oxidase is activated by the tryptophan metabolites 3-hydroxyanthranilate and 3-hydroxykynurenine. 3-Hydroxyanthranilate at concentrations of 0.03 mM relieves the inhibition elicited by accumulation of the substrate pyridoxine-5-P (Ki = 60 microM). The results of fluorometric measurements indicate that four molecules of 3-hydroxyanthranilate bind to the dimeric enzyme (56 kDa) with an association constant of 5.5 x 10(4) M-1. Differential spectral measurements failed to detect any direct interaction between the cofactor FMN and the effector 3-hydroxyanthranilate. These results are consistent with the hypothesis that the effector molecules bind to sites of the dimeric protein distinct from the cofactor site. Limited chymotrypsin digestion of pyridoxine-5-P oxidase yields catalytically active species that are no longer susceptible to activation by 3-hydroxykynurenine. A polypeptide of 16 kDa containing FMN and endowed with full catalytic activity was isolated by ion-exchange chromatography. It is postulated that the structural domain associated with catalytic activity composes approximately one-half of the molecular mass of pyridoxine-5-P oxidase (28 kDa), whereas the remaining portion of the macromolecule contains regulatory binding sites.
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PMID:Catalytic and regulatory properties of native and chymotrypsin-treated pyridoxine-5-phosphate oxidase. 193 34

The brewer's yeast old yellow enzyme (OYE) was reconstituted with 8-fluoro-8-demethyl FMN (8F-FMN). The reconstituted enzyme exhibited absorption maxima at 355 and 450 nm in the visible region. This reconstituted enzyme underwent no further spectral changes, showing no evidence of modification in the flavin moiety. However, when the reconstituted enzyme was subjected to specific limited proteolysis with bovine alpha-chymotrypsin, gradual spectral changes were observed with disappearance of the 355- and 450-nm bands accompanied by the appearance of a new band at 496 nm. Identical spectral changes were observed when the proteolytically cleaved OYE (nicked OYE) was reconstituted with 8F-FMN. The process associated with these spectral changes was found to be unimolecular by kinetic analysis. Reverse-phase HPLC analysis revealed that these spectral changes resulted from covalent bond formation between 8F-FMN and the protein moiety after the proteolytic cleavage of the protein into 14K and 34K fragments. The reverse-phase HPLC monitored at 490 nm showed that the chromophore with 496 nm absorption maximum was covalently attached to the 14K fragment. The amino acid sequence analysis of the flavinylated 14K fragment together with that of the 14K fragment of native OYE indicated that the N-terminal leucine of the 14K fragment is the site of flavinylation. These findings imply that the amino group of the N-terminal leucine of the 14K fragment became available as the result of proteolysis and that this amino group nucleophilically attacked the 8-position of 8F-FMN, forming a covalent bond between the flavin moiety and the 14K fragment.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Studies on the flavin-binding region of old yellow enzyme with an active site probe, 8-fluoro-8-demethyl FMN. 201 74

Limited proteolysis of brewer's yeast old yellow enzyme (OYE) was carried out with bovine pancreatic alpha-chymotrypsin. The reaction proceeded with a decrease of the NADPH oxidase activity, generating specifically two peptides (designated as 34K and 14K fragments) with apparent molecular weights of 34,000 and 14,000, respectively. The same proteolytic treatment of apo OYE resulted in rapid and complete digestion of the protein. The 34K and 14K fragments are so intimately associated with each other that the isolation of each peptide from the other in the native form was unsuccessful. However, the complex of the two fragments was separated from the intact OYE and termed "nicked OYE." Nicked OYE still retained FMN and showed a visible-absorption spectrum slightly modified from that of intact OYE. Nicked OYE showed decreased affinity toward rho-bromophenol as compared to intact OYE. Nicked OYE exhibited lower Km and Vmax values than intact OYE in the NADPH oxidase reaction. The 34K and 14K fragments could be separated from each other by reversed-phase HPLC under denaturing conditions and the amino acid sequences of the two fragments and intact OYE in the amino terminal regions were determined. The N-terminal sequence of the 34K fragment coincided with that of intact OYE, indicating that the 34K fragment lies in the N-terminal side of OYE. The N-terminal sequence of the 14K fragment was found to show homology with the site of flavodoxin where it forms an electron-transfer complex with cytochrome c. The characteristic feature of this region is the presence of acidic residues and is shared by the FMN domain of NADPH-cytochrome P-450 reductase. We interpret these findings as indicating that OYE has a physiological role as an electron transfer component.
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PMID:On the structure of old yellow enzyme studied by specific limited proteolysis. 312 66

Dimethylglycine dehydrogenase (EC 1.5.99.2) and sarcosine dehydrogenase (EC 1.5.99.1) are the folate binding proteins of rat liver mitochondria. These two enzymes contain covalently bound flavin and catalyze similar oxidative demethylation reactions (Wittwer, A. J., and Wagner, C. (1981) J. Biol. Chem. 256, 4102-4108). Flavin-peptides have been purified from these two enzymes after proteolytic digestion by trypsin and chymotrypsin. The spectral and chromatographic properties of these flavin peptides changed after treatment with nucleotide pyrophosphatase in a manner consistent with the conversion of an FAD-peptide to an FMN-peptide. The pKa for pH-dependent fluorescence quenching of the purified flavin-peptides was not affected by borohydride reduction which, in conjunction with the pKa values, indicated that the flavin was covalently linked via the 8 alpha position of the isoalloxazine ring to an imidazole N(3) of a histidine residue. Peptides from both enzymes showed histidylflavin at the N terminus. Amino acid composition and sequence analysis showed that the flavin-peptide from dimethylglycine dehydrogenase was His(flavin)-Ala-Ala-Gly-Leu. Amino acid composition and N-terminal analysis suggested the sequence of the flavin-peptide of sarcosine dehydrogenase was His(flavin)-(Ala, Gly,Thr)-Leu.
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PMID:Identification of the covalently bound flavin of dimethylglycine dehydrogenase and sarcosine dehydrogenase from rat liver mitochondria. 649 Jun 27

Flavocytochrome b2 from baker's yeast is a bifunctional tetrameric protein which carries two prosthetic groups, FMN and heme, per subunit of Mr 58 000. The amino terminus of the subunit is wrapped around the heme and constitutes the so-called cytochrome b2 core (Mr 11 000), homologous to cytochrome b5. It has been shown in the past that a number of proteases (yeast proteases, chymotrypsin) preferentially cleave the peptide chain at a point situated much further down the polypeptide chain than the C terminus of the heme-binding domain. Some enzymatic parameters are concomitantly modified, but not the quaternary structure. This paper describes the conditions for selective proteolysis of intact flavocytochrome b2 and of its various previously studied stable nicked forms by the protease from Staphylococcus aureus V8. Successive attack by a combination of two proteases is also described. We have established the amino acid sequence of the area where proteolytic attack takes places, and shown that chymotrypsin and S. aureus protease open only one bond, whereas yeast proteases remove five residues from the central part. The various nicked forms, some of which have lost up to 16 amino acid residues, have been enzymatically characterized. These and previous results lend support to, but do not prove, the idea that the flavodehydrogenase part of flavocytochrome b2 may be composed of two domains, linked by the region accessible to proteases. That area might constitute a hinge or rather a clasp between the domains.
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PMID:Study of a zone highly sensitive to proteases in flavocytochrome b2 from Saccharomyces cerevisiae. 703 12

Wild-type flavocytochrome b2 (L-lactate dehydrogenase) from Saccharomyces cerevisiae, as well as a number of its point mutants, can be expressed to a reasonable level as recombinant proteins in Escherichia coli (20-25 mg per liter culture) with a full complement of prosthetic groups. At the same expression level, active-site mutants Y254L and D282N, on the other hand, were obtained with an FMN/heme ratio significantly less than unity, which could not be raised by addition of free FMN. Evidence is provided that the flavin deficit is due to incomplete prosthetic group incorporation during biosynthesis. Flavin-free and holo-forms for both mutants could be separated on a Blue-Trisacryl M column. The far-UV CD spectra of the two forms of each mutant protein were very similar to one another and to that of the wild-type enzyme, suggesting the existence of only local conformational differences between the active holo-enzymes and the nonreconstitutable flavin-free forms. Selective proteolysis with chymotrypsin attacked the same bond for the two mutant holo-enzymes as in the wild-type one, in the protease-sensitive loop. In contrast, for the flavin-free forms of both mutants, cleavage occurred at more than a single bond. Identification of the cleaved bonds suggested that the structural differences between the mutant flavin-free and holo-forms are located mostly at the C-terminal end of the barrel, which carries the prosthetic group and the active site. Altogether, these findings suggest that the two mutations induce an alteration of the protein-folding process during biosynthesis in E. coli; as a result, the synchrony between folding and flavin insertion is lost. Finally, a preliminary kinetic characterization of the mutant holo-forms showed the Km value for lactate to be little affected; kcat values fell by a factor of about 70 for the D282N mutant and of more than 500 for the Y254L mutant, compared to the wild-type enzyme.
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PMID:On the lack of coordination between protein folding and flavin insertion in Escherichia coli for flavocytochrome b2 mutant forms Y254L and D282N. 766 48

Nitric oxide synthase (EC 1.14.13.39) catalyses the conversion of arginine, NADPH and oxygen to nitric oxide and citrulline, using haem, (6R)-5,6,7,8-tetrahydro-l-biopterin (tetrahydrobiopterin), calmodulin, FAD and FMN as cofactors. The enzyme consists of a central calmodulin-binding sequence flanked on the N-terminal side by a haem-binding region that contains the arginine and tetrahydrobiopterin sites and on the C-terminal side by a region homologous with NADPH:cytochrome P-450 reductase. By using domain boundaries defined by limited proteolysis of full-length enzyme, recombinant haem-binding regions of rat brain neuronal nitric oxide synthase were expressed and purified. Two proteins were made in high yield: one, corresponding to residues 221-724, contained bound haem and tetrahydrobiopterin and was able to bind Nomega-nitro-l-arginine (nitroarginine) or arginine; the other, containing residues 350-724, contained bound haem but was unable to bind tetrahydrobiopterin, nitroarginine or arginine. These results showed that rat brain neuronal nitric oxide synthase contains a critical determinant for arginine/tetrahydrobiopterin binding between residues 221 and 350. Limited proteolysis with chymotrypsin of the former protein resulted in a new species with an N-terminal residue 275 that retained the ability to bind nitroarginine, further defining the critical region for arginine binding as being between 275 and 350. Comparison of the sequences of nitric oxide synthase and the tetrahydrobiopterin-requiring amino acid hydroxylases revealed a similarity in the region between residues 470 and 600, suggesting that this might represent the core region of the pterin-binding site. The stoichiometries of binding of substrate and cofactors to the recombinant domains were not more than 0.5 mol/mol of monomer, suggesting that there might be a single high-affinity site per dimer.
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PMID:Delineation of the arginine- and tetrahydrobiopterin-binding sites of neuronal nitric oxide synthase. 917 72

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

Riboflavin (RF) upon irradiation with fluorescent light generates reactive oxygen species like superoxide anion, singlet and triplet oxygen, flavin radicals and substantial amounts of hydrogen peroxide (H2O2). H2O2 can freely penetrate cell membrane and react with a transition metal ion like Cu(ll), generating hydroxyl radical via the modified metal-catalyzed Haber-Weiss reaction. Earlier, it was reported that trypsin-chymotrypsin mixture served as an indirect antioxidant and decreased free radical generation. Thus, in the present study, we used photoilluminated RF as a source of ROS to investigate the effect of free radicals on the activity of trypsin. We also compared the damaging effect of photoilluminated RF and RF-Cu(ll) system using trypsin as a target molecule. RF caused fragmentation of trypsin and the effect was further enhanced, when Cu(II) was added to the reaction. Results obtained with various ROS scavengers suggested that superoxide radical, singlet and triplet oxygen were predominantly responsible for trypsin damage caused by photoilluminated RF. On the other hand, when Cu(ll) was added to the reaction, hydroxyl radical was mainly responsible for trypsin damage. A mechanism of generation of various ROS in the reaction is also proposed. Trypsin did not show any antioxidant effect with RF alone or with RF-Cu(II) combination.
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PMID:Photoilluminated riboflavin/riboflavin-Cu(II) inactivates trypsin: Cu(II) tilts the balance. 1713 39


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