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)

Previous experiments in our laboratory with Saccharomyces cervisiae flavocytochrom b2 indicated that both fragments alpha and beta of the enzyme after cleavage by yeast proteases are required to form the flavin site. More detailed experiments have not been carried out on the nicked Hansenula anomala enzyme obtained by tryptic cleavage. A method has been devised that gives a quantitative separation in 4 M urea of beta, and alpha with its heme still bound. The characteristics of the various species: isolated alpha and beta and mixed alpha + beta were studied in 4 M urea and after elimination of this reagent by dialysis in the presence of FMN and 2-mercaptoethanol. Several methods, including heme spectroscopy, tryptophan fluorescence, sedimentation studies, and titration of bound flavin, were used. The results indicate that isolated alpha and beta have a folded globular structure after renaturation. The flavin binding to the alpha + beta mixture was important (50-100%) with recovery of the flavodehydrogenase activity. In contrast, binding was not detectable (< 0.5%, Kf > 10 mM) for isolated alpha and beta. As far as mononucleotide binding is concerned, such a cooperative requirement for two folding domains has never been reported in other enzymes. The present results are discussed together with others obtained in our laboratory which demonstrate that, as deduced from their sensitivity to trypsin, the structure of S. cerevisiae and H. anomala flavocytochrome b2 protomers is triglobular 'n-x-beta' (n and x combined within alpha). The tetramer assembly, which remains intact as a nicked enzyme (alpha beta)4 after the first trypsin cleavage, is broken down following a second cleavage of the chain into four cytochrome cores (n) and a functional T-flavodehydrogenase entity, a tetramer of the type (x beta)4.
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PMID:A flavin-mononucleotide-binding site in Hansenula anomala nicked flavocytochrome b2, requiring the association of two domains. 743 81

Nitric oxide synthase (NOS) catalyzes the NADPH-dependent, Ca2+/calmodulin-dependent formation of NO and citrulline from L-arginine and molecular oxygen. The localization of the heme-binding consensus sequence in the NH2-terminal half of NOS and of the binding sequences for nucleotides (FMN and FAD) in the COOH-terminal half suggests a bidomain structure. In addition, the presence of a putative calmodulin-binding sequence between the heme- and flavin-binding domains of the enzyme suggests a role for calmodulin in modulating a spatial orientation of these domains that is required for catalytic activity. First, to determine the effects of calmodulin and the functionality of the separated domains, Ca2+/calmodulin binding-induced conformational changes in NOS were measured by fluorescence quenching, from which a binding constant of approximately 1 nM for calmodulin was calculated. Second, electron transport to various artificial acceptors was measured. The addition of Ca2+/calmodulin increased cytochrome c reduction from 10-15-fold while stimulating the rate of 2,6-dichlorophenolindophenol and ferricyanide reduction only slightly, if at all. Calmodulin stimulation of NOS results in NADPH-mediated cytochrome c reduction, which is sensitive to superoxide dismutase, and the reduction of acetylated cytochrome c, which is only weakly reducible by unstimulated NOS. Thus, this stimulated activity is presumably superoxide anion-mediated. Third, limited proteolysis of NOS in the absence of calmodulin resulted in a time-dependent increase in cytochrome c reductase activity, which was not inhibitable by superoxide dismutase, and a decrease in catalysis of NO formation. SDS-polyacrylamide gel electrophoresis analysis of the tryptic digest demonstrated the formation of approximately 89- and approximately 79-kDa fragments. Sequence analysis of the peptides confirmed that trypsin cleaves the enzyme in the putative calmodulin-binding region beginning with Ala728. This region was protected from proteolysis by the addition of Ca2+/calmodulin. The separated NH2-terminal domain exhibited the characteristic spectrum of bound heme, while the COOH-terminal domain showed the characteristic spectrum of bound flavins. Other cleavage patterns were obtained in the presence of calmodulin. The data demonstrate that the heme- and flavin-binding domains of NOS can be isolated in functionally intact forms.
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PMID:Evidence for a bidomain structure of constitutive cerebellar nitric oxide synthase. 751 50

Macrophage NO synthase (NOS) is a dimeric enzyme comprising two identical 130 kDa subunits and contains iron protoporphyrin IX (heme), tetrahydrobiopterin, FAD, FMN, and calmodulin. We have carried out limited proteolysis to locate the domains involved in prosthetic group binding and subunit interaction. Trypsin cleaved the subunits of dimeric macrophage NOS at a single locus, splitting the enzyme into two fragments whose denatured molecular masses were 56 and 74 kDa. The smaller fragments remained dimeric in their native form (112 kDa), contained heme and tetrahydrobiopterin, and could bind L-arginine, CO, or imidazole. In contrast, the larger fragments were monomeric in their native form, contained FAD, FMN, and CAM, and bound NADPH. Although neither purified fragment alone or in combination catalyzed NO synthesis from L-arginine, the flavin-containing fragment did catalyze cytochrome c reduction at a rate that was equivalent to that of native dimeric NOS. These results indicate that trypsin cuts macrophage NOS into two domains that can exist and function independently of one another. The domain that binds heme, H4biopterin, and substrate is also responsible for maintaining the NOS dimeric structure, while the domain containing FAD, FMN, and CAM is not required for subunit interaction. This suggests a structural model for macrophage NOS in which the subunits align in a head-to-head manner, with the oxygenase domains interacting to form a dimer and the reductase domains existing as independent extensions.
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PMID:Macrophage NO synthase: characterization of isolated oxygenase and reductase domains reveals a head-to-head subunit interaction. 753 45

High levels of conversion of 14C-labelled pristinamycin IIB (PIIB) to pristinamycin IIA (PIIA) were obtained in vivo in Streptomyces pristinaespiralis and in some other streptogramin A producers. This established that PIIB was an intermediate on the pathway to PIIA. In addition, in vitro studies with cell-free protein preparations demonstrated that the oxidation of PIIB to PIIA is a complex process requiring NADH, riboflavin 5'-phosphate (FMN), and molecular oxygen. Two enzymes were shown to be necessary to catalyze this reaction. Both were purified to homogeneity from S. pristinaespiralis by a coupled enzyme assay based on the formation of PIIA and by requiring addition of the complementing enzyme. One enzyme was purified about 3,000-fold by a procedure including a decisive affinity chromatography step on FMN-agarose. It was shown to be a NADH:FMN oxidoreductase (E.C. 1.6.8.1.) (hereafter called FMN reductase), providing reduced FMN (FMNH2) to the more abundant second enzyme. The latter was purified only 160-fold and was called PIIA synthase. Our data strongly suggest that this enzyme catalyzes a transient hydroxylation of PIIB by molecular oxygen immediately followed by a dehydration leading to PIIA. The native PIIA synthase consists of two different subunits with Mrs of around 50,000 and 35,000, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, while the FMN reductase seems to be a monomer with a Mr of around 28,000 and containing one molecule of tightly bound FMN. Stepwise Edman degradation of the entire polypeptides or some of their trypsin-digested fragments provided amino acid sequences for the two isolated proteins.
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PMID:Purification of the two-enzyme system catalyzing the oxidation of the D-proline residue of pristinamycin IIB during the last step of pristinamycin IIA biosynthesis. 766 8

The kinetics of tryptic proteolysis of rabbit skeletal muscle phosphorylase b has been registered by the diminishing of protein fluorescence intensity at lambda = 335 nm (excitation at 290 nm) or by the disappearance of the enzyme activity (0.02 M Hepes buffer, pH 6.8, 37 degrees C). The first procedure showed that flavins (riboflavin, FMN, FAD) protected the enzyme against tryptic digestion. Microscopic dissociation constants for the complexes of phosphorylase b with riboflavin, FMN and FAD were calculated from dependences of the initial digestion rate on the flavin concentration. They where equal to 30 +/- 1, 15.8 +/- 0.2 and 36 +/- 1 microM, respectively. No influence of FMN on the rate of the tryptic hydrolysis of phosphorylase b was observed when using the second procedure (enzyme activity test). FMN completely prevents the formation of 69-, 81- and 85-kDa fragments during 20 min incubation of phosphorylase b with trypsin.
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PMID:Effect of flavins on the rate of proteolytic digestion of muscle glycogen phosphorylase b. 836 99

Long-chain L-alpha-hydroxy acid oxidase from rat kidney is a member of the family of FMN-dependent alpha-hydroxy-acid-oxidizing enzymes. With the knowledge of the recently determined amino acid sequence, the cDNA encoding the enzyme has now been cloned using the polymerase chain reaction. The 1648-bp cDNA contains an open reading frame coding for the 352 residues of the previously determined sequence, preceded by a methionine codon. In addition, several clones were found to present a nine-base insertion, predicting the existence of an isoform with a tripeptide VRK inserted between residues 188 and 189 of the mature protein. The presence of about 10% of this isoform in the oxidase purified from rat kidney was indeed identified by amino acid sequencing. A recombinant active enzyme was obtained as a protein fused to glutathione S-transferase using the bacterial expression plasmid pGEX-3X. Physico-chemical characterization indicated, for the fused enzyme, properties similar to those of the rat kidney protein. When the chimaera was submitted to factor Xa, proteolysis at the engineered cleavage point was poor. Separation of hydroxy acid oxidase from glutathione S-transferase could not be achieved with trypsin either. With both proteases, the initial cleavage point appeared to be in a peptide loop internal to the hydroxy acid oxidase sequence, close to or in the tripeptide insertion locus and not at the engineered factor-Xa-cleavage point. Comparative tryptic proteolysis of the rat kidney enzyme yielded a form cleaved in the same loop.
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PMID:Molecular cloning and nucleotide sequence of cDNA encoding rat kidney long-chain L-2-hydroxy acid oxidase. Expression of the catalytically active recombinant protein as a chimaera. 850 89

Nitric oxide synthase (EC 1.14.13.39) binds arginine and NADPH as substrates, and FAD, FMN, tetrahydrobiopterin, haem and calmodulin as cofactors. The protein consists of a central calmodulin-binding sequence flanked on the N-terminal side by a haem-binding region, analogous to cytochrome P-450, and on the C-terminal side by a region homologous with NADPH:cytochrome P-450 reductase. The structure of recombinant rat brain nitric oxide synthase was analysed by limited proteolyis. The products were identified by using antibodies to defined sequences, and by N-terminal sequencing. Low concentrations of trypsin produced three fragments, similar to those in a previous report [Sheta, McMillan and Masters (1994) J. Biol. Chem. 269, 15147-15153]: that of Mr approx. 135000 (N-terminus Gly-221) resulted from loss of the N-terminal extension (residues 1-220) unique to neuronal nitric oxide synthase. The fragments of Mr 90000 (haem region) and 80000 (reductase region, N-terminus Ala-728) were produced by cleavage within the calmodulin-binding region. With more extensive trypsin treatment, these species were shown to be transient, and three smaller, highly stable fragments of Mr 14000 (N-terminus Leu-744 within the calmodulin region), 60000 (N-terminus Gly-221) and 63000 (N-terminus Lys-856 within the FMN domain) were formed. The species of Mr approx. 60000 represents a domain retaining haem and nitroarginine binding. The two species of Mr 63000 and 14000 remain associated as a complex. This complex retains cytochrome c reductase activity, and thus is the complete reductase region, yet cleaved at Lys-856. This cleavage occurs within a sequence insertion relative to the FMN domain present in inducible nitric oxide synthase. Prolonged proteolysis treatment led to the production of a protein of Mr approx. 53000 (N-terminus Ala-953), corresponding to a cleavage between the FMN and FAD domains. The major products after chymotryptic digestion were similar to those with trypsin, although the pathway of intermediates differed. The haem domain was smaller, starting at residue 275, yet still retained the arginine binding site. These data have allowed us to identify stable domains representing both the arginine/haem-binding and the reductase regions.
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PMID:Identification of the domains of neuronal nitric oxide synthase by limited proteolysis. 866 Mar 10

It has been shown that treatment of bovine mitochondrial complex I (NADH-ubiquinone oxidoreductase) with NADH or NADPH, but not with NAD or NADP, increases the susceptibility of a number of subunits to tryptic degradation. This increased susceptibility involved subunits that contain electron carriers, such as FMN and iron-sulfur clusters, as well as subunits that lack electron carriers. Results shown elsewhere on changes in the cross-linking pattern of complex I subunits when the enzyme was pretreated with NADH or NADPH (Belogrudov, G., and Hatefi, Y. (1994) Biochemistry 33, 4571-4576) also indicated that complex I undergoes extensive conformation changes when reduced by substrate. Furthermore, we had previously shown that in submitochondrial particles the affinity of complex I for NAD increases by >/=20-fold in electron transfer from succinate to NAD when the particles are energized by ATP hydrolysis. Together, these results suggest that energy coupling in complex I may involve protein conformation changes as a key step. In addition, it has been shown here that treatment of complex I with trypsin in the presence of NADPH, but not NADH or NAD(P), produced from the 39-kDa subunit a 33-kDa degradation product that resisted further hydrolysis. Like the 39-kDa subunit, the 33-kDa product bound to a NADP-agarose affinity column, and could be eluted with a buffer containing NADPH. It is possible that together with the acyl carrier protein of complex I the NADP(H)-binding 39-kDa subunit is involved in intramitochondrial fatty acid synthesis.
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PMID:Mitochondrial NADH-ubiquinone oxidoreductase (Complex I). Effect of substrates on the fragmentation of subunits by trypsin. 952 11

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 Arg107 of the alpha subunit is a conserved residue for all known bacterial luciferases. The phosphate moiety of the reduced flavin mononucleotide (FMNH(2)) side chain has been hypothesized to be anchored at this site (A. J. Fisher, F. M. Raushel, T. O. Baldwin, and I. Rayment Biochemistry 34, 6581-6586, 1995). Mutations of alphaArg107 of the Vibrio harveyi luciferase to alanine, serine, and glutamate were carried out to test such a hypothesis. These variants were characterized and compared with the wild-type luciferase with respect to their K(m) for decanal, FMNH(2), and reduced riboflavin in both low- (0.01 or 0.05 M) and high- (0.3 M) phosphate buffers at pH 7.0. Results are consistent with the hypothesized binding of the FMNH(2) phosphate group by alphaArg107. Moreover, the alphaArg107 residue was apparently important in the expression of the luciferase maximal activity and aldehyde binding. Phosphate ion is also known to have other effects on luciferase stability. We compared the three luciferase variants with the native enzyme with respect to the decay rate of the FMN 4a-hydroperoxide intermediate II, and rates of inactivation by trypsin digestion, modification by N-ethylmaleimide, and heat treatment in low- and high-phosphate buffers. On the basis of patterns of the phosphate effects, alphaArg107 appeared to be important to the enhancement of luciferase stability against trypsin proteolysis at high phosphate but was not involved in regulating the intermediate II decay or sensitivity to N-ethylmaleimide modification. Differential effects of mutations on luciferase thermal stability were observed. It is uncertain whether alphaArg107 is involved in the enhanced thermal stability of the native luciferase in high phosphate buffer.
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PMID:Relationship between the conserved alpha subunit arginine 107 and effects of phosphate on the activity and stability of Vibrio harveyi luciferase. 1049 75


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