Tryptophan Fluorescence of the lux-SpecificVibrio harveyiAcyl-ACP Thioesterase and Its Tryptophan Mutants:  Structural Properties and Ligand-Induced Conformational Change†

Biochemistry ◽  
1998 ◽  
Vol 37 (46) ◽  
pp. 16130-16138 ◽  
Author(s):  
Jun Li ◽  
Rose Szittner ◽  
Edward A. Meighen
Keyword(s):  
Conformational Change ◽  
Structural Properties ◽  
Tryptophan Fluorescence

scholarly journals Conformation-dependent inactivation of human betaine-homocysteine S-methyltransferase by hydrogen peroxide in vitro

2005 ◽  
Vol 392 (3) ◽  
pp. 443-448 ◽  
Author(s):  
Catherine M. Miller ◽  
Sandra S. Szegedi ◽  
Timothy A. Garrow
Keyword(s):  
Hydrogen Peroxide ◽  
Binding Site ◽  
Conformational Change ◽  
Binding Sites ◽  
Quaternary Structure ◽  
Substrate Binding ◽  
Tryptophan Fluorescence ◽  
H2o2 Treatment ◽  
Oxidative Inactivation

Betaine-homocysteine S-methyltransferase (BHMT) transfers a methyl group from betaine to Hcy to form DMG (dimethylglycine) and Met. The reaction is ordered Bi Bi; Hcy is the first substrate to bind and Met is the last product off. Using intrinsic tryptophan fluorescence [Castro, Gratson, Evans, Jiracek, Collinsova, Ludwig and Garrow (2004) Biochemistry 43, 5341–5351], it was shown that BHMT exists in three steady-state conformations: enzyme alone, enzyme plus occupancy at the first substrate-binding site (Hcy or Met), or enzyme plus occupancy at both substrate-binding sites (Hcy plus betaine, or Hcy plus DMG). Betaine or DMG alone do not bind to the enzyme, indicating that the conformational change associated with Hcy binding creates the betaine-binding site. CBHcy [S-(δ-carboxybutyl)-D,L-homocysteine] is a bisubstrate analogue that causes BHMT to adopt the same conformation as the ternary complexes. We report that BHMT is susceptible to conformation-dependent oxidative inactivation. Two oxidants, MMTS (methyl methanethiosulphonate) and hydrogen peroxide (H2O2), cause a loss of the enzyme's catalytic Zn (Zn2+ ion) and a correlative loss of activity. Addition of 2-mercaptoethanol and exogenous Zn after MMTS treatment restores activity, but oxidation due to H2O2 is irreversible. CD and glutaraldehyde cross-linking indicate that H2O2 treatment causes small perturbations in secondary structure but no change in quaternary structure. Oxidation is attenuated when both binding sites are occupied by CBHcy, but Met alone has no effect. Partial digestion of ligand-free BHMT with trypsin produces two large peptides, excising a seven-residue peptide within loop L2. CBHcy but not Met binding slows down proteolysis by trypsin. These findings suggest that L2 is involved in the conformational change associated with occupancy at the betaine-binding site and that this conformational change and/or occupancy at both ligand-binding sites protect the enzyme from oxidative inactivation.

scholarly journals The conformational changes of α2-macroglobulin induced by methylamine or trypsin. Characterization by extrinsic and intrinsic spectroscopic probes

1987 ◽  
Vol 243 (1) ◽  
pp. 47-54 ◽  
Author(s):  
L J Larsson ◽  
P Lindahl ◽  
C Hallén-Sandgren ◽  
I Björk
Keyword(s):  
Conformational Change ◽  
Conformational Changes ◽  
Fluorescence Emission ◽  
Tryptophan Fluorescence ◽  
Cross Linking ◽  
Bait Region ◽  
Close Proximity ◽  
Tryptophan Residues ◽  
Thioester Bond ◽  
Bond Region

The conformational changes around the thioester-bond region of human or bovine alpha 2M (alpha 2-macroglobulin) on reaction with methylamine or trypsin were studied with the probe AEDANS [N-(acetylaminoethyl)-8-naphthylamine-1-sulphonic acid], bound to the liberated thiol groups. The binding affected the fluorescence emission and lifetime of the probe in a manner indicating that the thioester-bond region is partially buried in all forms of the inhibitor. In human alpha 2M these effects were greater for the trypsin-treated than for the methylamine-treated inhibitor, which both have undergone similar, major, conformational changes. This difference may thus be due to a close proximity of the thioester region to the bound proteinase. Reaction of trypsin with thiol-labelled methylamine-treated bovine alpha 2M, which retains a near-native conformation and inhibitory activity, indicated that the major conformational change accompanying the binding of proteinases involves transfer of the thioester-bond region to a more polar environment without increasing the exposure of this region at the surface of the protein. Labelling of the transglutaminase cross-linking site of human alpha 2M with dansylcadaverine [N-(5-aminopentyl)-5-dimethylaminonaphthalene-1-sulphonamide] suggested that this site is in moderately hydrophobic surroundings. Reaction of the labelled inhibitor with methylamine or trypsin produced fluorescence changes consistent with further burial of the cross-linking site. These changes were more pronounced for trypsin-treated than for methylamine-treated alpha 2M, presumably an effect of the cleavage of the adjacent ‘bait’ region. Solvent perturbation of the u.v. absorption and iodide quenching of the tryptophan fluorescence of human alpha 2M showed that one or two tryptophan residues in each alpha 2M monomer are buried on reaction with methylamine or trypsin, with no discernible change in the exposure of tyrosine residues. Together, these results indicate an extensive conformational change of alpha 2M on reaction with amines or proteinases and are consistent with several aspects of a recently proposed model of alpha 2M structure [Feldman, Gonias & Pizzo (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 5700-5704].

scholarly journals Impairment of the catalytic activity of Escherichia coli ribonucleic acid polymerase by a unique reaction of 4-chloro-7-nitrobenzofurazan

1979 ◽  
Vol 183 (2) ◽  
pp. 255-268 ◽  
Author(s):  
S C Bratcher ◽  
K Nitta ◽  
M J Kronman
Keyword(s):  
Escherichia Coli ◽  
Catalytic Activity ◽  
Conformational Change ◽  
Active Site ◽  
Tryptophan Fluorescence ◽  
Chain Elongation ◽  
Nitrobenzoic Acid ◽  
Unique Reaction ◽  
Calf Thymus ◽  
Partial Inactivation

Escherichia coli RNA polymerase loses 55-65% of its catalytic activity on reaction with Nbf-Cl (4-choro-7-nitrobenzofurazan). This partial inactivation was shown to be the result of specific impairment of RNA-chain elongation, since initiation of RNA chains was not altered after treatment with Nbf-Cl. The site of reaction was shown to be a unique thiol on the beta-subunit. This thiol is not accessible to reaction with 5,5′-dithiobis-(2-nitrobenzoic acid). No protection of the enzyme against reaction with Nbf-Cl could be obtained with the inhibitor rifamycin nor with calf thymus DNA, GTP or 1,10-phenanthroline, indicating that the unique thiol is probably not within the active site. The specific impairment of RNA-chain elongation thus appears to be the result of a local conformational change which leaves chain initiation unimpaired. Changes observed in the tryptophan fluorescence spectrum of the enzyme or reaction with Nbf-Cl are consistent with formation of a Meisenheimer complex of the reagent with a nucleophilic group on the enzyme near the reactive thiol. It is proposed that formation of such a complex and a subsequent conformational change renders this thiol unusually susceptible to reaction with Nbf-Cl.

scholarly journals Kinetics of Light-Induced Conformational Change in Phot1 LOV2-Jalpha Probed By Transient Tryptophan Fluorescence

2010 ◽  
Vol 98 (3) ◽  
pp. 234a
Author(s):  
Daniel Hoersch ◽  
Farzin Bolourchian ◽  
Harald Otto ◽  
Maarten P. Heyn ◽  
Roberto A. Bogomolni
Keyword(s):  
Conformational Change ◽  
Tryptophan Fluorescence ◽  
Kinetics Of
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