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)

Escherichia coli RH1 contains a mutation causing complete loss of pyridine nucleotide transhydrogenase activity. A single base change in the chromosomal DNA resulted in the replacement of Gly314 of the beta subunit by a Glu residue. The mutant enzyme was partially purified and its trypsin cleavage products examined. The distinct pattern of polypeptides given by proteolysis of the normal transhydrogenase in the presence of NADP(H) was absent when the mutant enzyme was treated with trypsin. However, the beta subunit of the mutant enzyme retained its ability to bind to NAD-agarose. Further substitutions were made at Gly314 converting it to Ala, Val or Cys by the use of site-directed mutagenesis. All substitutions for Gly314 abolished the activity completely. The enzyme containing the Gly314----Ala mutation was studied in detail and behaved exactly as the enzyme containing the Gly314----Glu mutation. It is concluded that the mutation in the beta subunit abolished the NADP(H)-induced conformational change in the mutant enzyme. This conformational change, caused by NADP(H) binding, is required to cleave the normal beta subunit at Arg265 by trypsin. The genes encoding the pyridine nucleotide transhydrogenase were completely resequenced and several corrections have been made to the previously published sequence [Clarke et al. (1986) Eur. J. Biochem. 158, 647-653].
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PMID:A mutation at Gly314 of the beta subunit of the Escherichia coli pyridine nucleotide transhydrogenase abolishes activity and affects the NADP(H)-induced conformational change. 163 24

The mitochondrial proton-translocating nicotinamide nucleotide transhydrogenase is embedded in the inner membrane as a homodimer of monomer Mr = 109,288. Its N-terminal 430 residues and C-terminal 200 residues protrude into the matrix, whereas its central 400 residues appear to intercalate into the inner membrane as 14 hydrophobic clusters of about 20 residues each (Yamaguchi, M., and Hatefi, Y. (1991) J. Biol. Chem. 266, 5728-5735). Treatment of mitoplasts (mitochondria denuded of outer membrane) with several proteolytic enzymes cleaves the transhydrogenase into a 72-kDa N-terminal and a 37-kDa C-terminal fragment. The cleavage site of proteinase K was determined to be Ala690-Ala691, which is located in a small loop of the transhydrogenase exposed on the cytosolic side of the inner membrane. This paper shows that the bisected transhydrogenase can be purified from proteinase K-treated mitoplasts with retention of greater than or equal to 85% transhydrogenase activity. The inactivation rate of the bisected enzyme by trypsin and N-ethylmaleimide was altered in the presence of NADP and NADPH, suggesting substrate-induced conformation changes similar to those reported previously for the intact transhydrogenase. Also, like the intact enzyme, proteoliposomes of the bisected transhydrogenase were capable of membrane potential formation and internal acidification coupled to NADPH----NAD transhydrogenation. The properties of the bisected transhydrogenase have been discussed in relation to those of the two-subunit Escherichia coli transhydrogenase, the bisected lac permease (via gene restriction), and the fragmented and reconstituted bacteriorhodopsin.
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PMID:Mitochondrial energy-transducing nicotinamide nucleotide transhydrogenase. Purification and properties of the proteinase K-bisected enzyme. 165 21

Fluorescein 5'-isothiocyanate binds almost selectively at the active site of lamb liver NADP-dependent 6-phosphogluconate dehydrogenase causing the inactivation of the enzyme. The substrate and the coenzyme protect against the loss of catalytic activity. The enzyme derivative was digested with trypsin, the labelled peptide was isolated by h.p.l.c. and its amino acid analysis allowed to establish that the inactivator binds to lysine 166 at the active site of the protein.
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PMID:Identification of the lysine residue involved in the inactivation of lamb liver 6-phosphogluconate dehydrogenase by fluorescein 5'-isothiocyanate. 181 97

Pig heart NADP-specific isocitrate dehydrogenase is inactivated by N-ethylmaleimide (NEM) (Colman, R. F., and Chu, R. (1970) J. Biol. Chem. 245, 601-607), and is completely protected against inactivation, but not against the incorporation of NEM, by isocitrate plus Mn2+. We have now treated the enzyme with [3H]NEM in the absence and presence of isocitrate plus Mn2+, digested it with trypsin, and isolated and sequenced the labeled Cys peptides. In the inactive enzyme, two major peptides, SSGGFVWACK and DLAGCIHGLSNVK, and two minor peptides, CATITPDEAR and EPIICK, were labeled at Cys. Upon reaction with [3H]NEM in the presence of isocitrate plus Mn2+, full catalytic activity was retained and only DLAGCIHGLSNVK was labeled; the Cys of this peptide is therefore not essential for catalysis. The modification of SSGGFVWACK appears to be the major cause of inactivation by NEM. The Cys in SSGGFVWACK may have a catalytic role, most likely in the strengthened binding of Mn2+ in the presence of isocitrate. Isocitrate dehydrogenase was carboxymethylated under denaturing conditions with [14C]iodoacetate and digested with trypsin; 6 unique labeled Cys peptides, containing 6 unique Cys residues, were purified and sequenced. Six corresponding peptides were isolated from enzyme treated under denaturing conditions with [3H]NEM. These results eliminate the previous uncertainty regarding the number of Cys residues in the enzyme. A comparison of the sequences of the NH2-terminal 30 residues and the 6 Cys peptides of the pig heart NADP-dependent isocitrate dehydrogenase with the Escherichia coli NADP enzyme provides evidence for great dissimilarity between the two enzymes.
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PMID:Cysteinyl peptides of pig heart NADP-dependent isocitrate dehydrogenase that are modified upon inactivation by N-ethylmaleimide. 186 31

Thioredoxin, reduced either enzymatically with NADPH and NADP-thioredoxin reductase or chemically with dithiothreitol, reduced alpha-amylase and trypsin inhibitor proteins from several sources. Included were cystine-rich seed representatives from wheat (alpha-amylase inhibitors), soybean (Bowman-Birk trypsin inhibitor), and corn (kernel trypsin inhibitor). This system also reduced other trypsin inhibitors: the soybean Kunitz inhibitor, bovine lung aprotinin, and egg white ovoinhibitor and ovomucoid proteins. The ability of these proteins to undergo reduction by thioredoxin was determined by 1) a coupled enzyme activation assay with chloroplast NADP-malate dehydrogenase or fructose-1,6-bisphosphatase, 2) a dye reduction assay with 5',5'-dithiobis(2-nitrobenzoic acid), and 3) a direct reduction method based on the fluorescent probe, monobromobimane, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Reduction experiments with the seed proteins were carried out with thioredoxin from wheat germ (h-type) or Escherichia coli; the corresponding experiments with the animal trypsin inhibitors were carried out with thioredoxin from calf thymus or E. coli. In all cases, thioredoxin appeared to act catalytically; the reduced form of glutathione was without effect. When considered in conjunction with earlier results on purothionin (confirmed and extended in the current study), the new findings suggest that the NADP/thioredoxin system functions in the reduction of protein inhibitors of seeds and animal tissues. These results also raise the question of the occurrence of glutaredoxin in plants, as E. coli glutaredoxin was found to promote the reduction of some but not all of the proteins tested.
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PMID:Role of the NADP/thioredoxin system in the reduction of alpha-amylase and trypsin inhibitor proteins. 187 51

The pyridine nucleotide transhydrogenase of Escherichia coli has an alpha 2 beta 2 structure (alpha: Mr, 54,000; beta: Mr, 48,700). Hydropathy analysis of the amino acid sequences suggested that the 10 kDa C-terminal portion of the alpha subunit and the N-terminal 20-25 kDa region of the beta subunit are composed of transmembranous alpha-helices. The topology of these subunits in the membrane was investigated using proteolytic enzymes. Trypsin digestion of everted cytoplasmic membrane vesicles released a 43 kDa polypeptide from the alpha subunit. The beta subunit was not susceptible to trypsin digestion. However, it was digested by proteinase K in everted vesicles. Both alpha and beta subunits were not attacked by trypsin and proteinase K in right-side out membrane vesicles. The beta subunit in the solubilized enzyme was only susceptible to digestion by trypsin if the substrates NADP(H) were present. NAD(H) did not affect digestion of the beta subunit. Digestion of the beta subunit of the membrane-bound enzyme by trypsin was not induced by NADP(H) unless the membranes had been previously stripped of extrinsic proteins by detergent. It is concluded that binding of NADP(H) induces a conformational change in the transhydrogenase. The location of the trypsin cleavage sites in the sequences of the alpha and beta subunits were determined by N- and C-terminal sequencing. A model is proposed in which the N-terminal 43 kDa region of the alpha subunit and the C-terminal 30 kDa region of the beta subunit are exposed on the cytoplasmic side of the inner membrane of E. coli. Binding sites for pyridine nucleotide coenzymes in these regions were suggested by affinity chromatography on NAD-agarose columns.
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PMID:Topological analysis of the pyridine nucleotide transhydrogenase of Escherichia coli using proteolytic enzymes. 193 78

Purified pea chloroplast NADP-malate dehydrogenase (S)-malate: NADP+ oxidoreductase, EC 1.1.1.82) was digested with trypsin and the resulting peptides were separated by HPLC and sequenced. Together with the information from earlier work (Fickenscher, K. et al. (1987) Eur. J. Biochem. 168, 653-658) the total sequence is not known to an extent of 78%. Comparison with the sequence of the corn NADP-malate dehydrogenase deduced from its cDNA (Metzler, M.C. et al. (1989) Plant Mol. Biol. 12, 713-722) showed 84% agreement; however, the 11 N-terminal residues exhibit only 27% similarity. The N- and C-terminal extrapeptides of the pea NADP-malate dehydrogenase when aligned with non-regulatory NAD-malate dehydrogenases from bacteria or mammals consist of 30 and 17 amino acids, respectively. Since all cysteine-containing peptides were sequenced, the number of eight cysteines per subunit of the pea enzyme was established. The native, oxidized enzyme is characterized by an extremely slow reactivity of two thiols. Titration of the thiols of the denatured, oxidized enzyme both with DTNB and with pCMB resulted in six thiols not involved in disulfide formation. Therefore, one disulfide bridge must be present per 38.9 kDa subunit. Analysis of disulfide bonds by urea gel electrophoresis confirmed this finding. Using digestion products of NADP-malate dehydrogenase with aminopeptidase K, the location of the single disulfide bridge was established to be on the N-terminal arm (Cys-12 and Cys-17) of the polypeptide chain.
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PMID:Primary structure and analysis of the location of the regulatory disulfide bond of pea chloroplast NADP-malate dehydrogenase. 198 82

A second thioredoxin, Ch1, distinct from the one recently reported [Decottignies, P., Schmitter, J.M., Jacquot, J. P., Dutka, S., Picaud, A. & Gadal, P. (1990) Arch, Biochem. Biophys. 280, 112-121] has been purified from the green alga, Chlamydomonas reinhardtii, and its functional and structural properties investigated. Its activity in various enzymatic assays has been compared with the activities of different plant thioredoxins (Ch2 from C. reinhardtii and spinach m and f). Ch1 cannot serve as a substrate for Escherichia coli thioredoxin reductase, but can be reduced by spinach ferredoxin-thioredoxin reductase. It is less efficient than its spinach counterpart in the activation of corn leaf NADP-dependent malate dehydrogenase by light or dithiothreitol, and it only activates spinach fructose-1,6-bisphosphatase at very high concentrations. The complete primary structure of C. reinhardtii thioredoxin Ch1 was determined by automated Edman degradation of the intact protein and of peptides derived from trypsin, chymotrypsin and Staphylococcus aureus V8 protease digestions. When needed, peptide masses were verified by plasma desorption mass spectrometry. Ch1 consists of a polypeptide of 111 amino acids (11634 Da) and contains the well-conserved active site sequence Trp-Cys-Gly-Pro-Cys. Compared to thioredoxins from other sources, the algal thioredoxin Ch1 displays few sequence similarities with all the thioredoxins sequenced so far. Preliminary evidence indicates that Ch1 may be an h-type thioredoxin.
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PMID:Characterization and primary structure of a second thioredoxin from the green alga, Chlamydomonas reinhardtii. 204 Mar 9

Two thioredoxins (named Ch1 and Ch2 in reference to their elution pattern on an anion-exchange column) have been purified to homogeneity from the green alga, Chlamydomonas reinhardtii. In this paper, we described the properties and the sequence of the most abundant form, Ch2. Its activity in various enzymatic assays has been compared with those of Escherichia coli and spinach thioredoxins. C. reinhardtii thioredoxin Ch2 can serve as a substrate for E. coli thioredoxin reductase with a lower efficiency when compared to the homologous system. In the presence of dithiothreitol (DTT), the protein is able to catalyze the reduction of porcine insulin. Thioredoxin Ch2 is as efficient as its spinach counterpart in the DTT or light activation of corn NADP-malate dehydrogenase, but it only activates spinach fructose-1, 6-bisphosphatase at very high concentrations. The complete primary structure of the C. reinhardtii thioredoxin Ch2 was determined by automated Edman degradation of the intact protein and of peptides derived from trypsin, chymotrypsin, clostripain, and SV8 protease digestions. It consists of a polypeptide of 106 amino acids (MW 11,808) and contains the well-conserved active site sequence Trp-Cys-Gly-Pro-Cys. The sequence of the algal thioredoxin Ch2 has been compared to that of thioredoxins from other sources and has the greatest similarity (67%) with the thioredoxin from Anabaena 7119.
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PMID:Purification, characterization, and complete amino acid sequence of a thioredoxin from a green alga, Chlamydomonas reinhardtii. 219 28

The mitochondrial nicotinamide nucleotide transhydrogenase catalyzes hydride ion transfer between NAD(H) and NADP(H) in a reaction that is coupled to proton translocation across the inner mitochondrial membrane. The enzyme (1043 residues) is composed of an N-terminal hydrophilic segment (approximately 400 residues long) which binds NAD(H), a C-terminal hydrophilic segment (approximately 200 residues long) which binds NADP(H), and a central hydrophobic segment (approximately 400 residues long) which appears to form about 14 membrane-intercalating clusters of approximately 20 residues each. Substrate modulation of transhydrogenase conformation appears to be intimately associated with its mechanism of proton translocation. Using trypsin as a probe of enzyme conformation change, we have shown that NADPH (and to a much lesser extent NADP) binding alters transhydrogenase conformation, resulting in increased susceptibility of several bonds to tryptic hydrolysis. NADH and NAD had little or no effect, and the NADPH concentration for half-maximal enhancement of trypsin sensitivity of transhydrogenase activity (35 microM) was close to the Km of the enzyme for NADPH. The NADPH-promoted trypsin cleavage sites were located 200-400 residues distant from the NADP(H) binding domain near the C-terminus. For example, NADPH binding greatly increased the trypsin sensitivity of the K410-T411 bond, which is separated from the NADP(H) binding domain by the 400-residue-long membrane-intercalating segment. It also enhanced the tryptic cleavage of the R602-L603 bond, which is located within the central hydrophobic segment. These results, which suggest a protein conformation change as a result of NADPH binding, have been discussed in relation to the mechanism of proton translocation by the transhydrogenase.
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PMID:Mitochondrial energy-linked nicotinamide nucleotide transhydrogenase: effect of substrates on the sensitivity of the enzyme to trypsin and identification of tryptic cleavage sites. 236 Nov 37


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