scholarly journals Chemical modification of glutamate dehydrogenase by 2,4,6-trinitrobenzenesulphonic acid

1969 ◽  
Vol 114 (3) ◽  
pp. 611-619 ◽  
Author(s):  
R. B. Freedman ◽  
G. K. Radda
Keyword(s):  
Chemical Modification ◽  
Glutamate Dehydrogenase ◽  
Enzymic Activity ◽  
Optical Rotatory Dispersion ◽  
Amino Group ◽  
Optical Rotatory ◽  
Amino Groups ◽  
Allosteric Effector ◽  
Structure Activity ◽  
Rapid Reaction

1. Modification with 2,4,6-trinitrobenzenesulphonic acid was studied for its effect on the structure, activity and response to regulatory effectors of ox liver glutamate dehydrogenase. 2. The modification affected amino groups only, and the relative reactivities of the amino groups of the enzyme are described. 3. A biphasic inactivation of the enzyme was observed and analysis of the course of inactivation and of modification showed that the rapid reaction of one amino group/subunit leads to loss of 80% of the enzymic activity. 4. NADH retarded the inactivation by 2,4,6-trinitrobenzenesulphonic acid, the protection increasing with NADH concentration. This, together with the previous observation, suggests that the rapidly reacting group is essential for the activity of the enzyme. 5. The effects of modification on the optical-rotatory-dispersion and sedimentation behaviour of the enzyme were studied. 6. The enzyme's response to the allosteric effector GTP was rapidly lost on modification, whereas its response to ADP was unaffected. Comparison of the inactivation and desensitization suggests that the reactive amino group is essential for both activity and GTP response, and that only a completely unmodified enzyme oligomer responds fully to GTP. 7. The merits of chemical-modification studies of large enzymes are discussed critically in connexion with the interpretation of these results.

scholarly journals Desensitization of glutamate dehydrogenase by reaction of tyrosine residues

1969 ◽  
Vol 114 (2) ◽  
pp. 419-427 ◽  
Author(s):  
N. C. Price ◽  
G. K. Radda
Keyword(s):  
Physical Properties ◽  
Glutamate Dehydrogenase ◽  
Sedimentation Coefficient ◽  
Enzymic Activity ◽  
Optical Rotatory Dispersion ◽  
Tyrosine Residue ◽  
Optical Rotatory ◽  
Chemical Modifications ◽  
Tyrosine Residues ◽  
Allosteric Inhibitor

1. The reaction of glutamate dehydrogenase with N-acetylimidazole and with tetranitromethane leads to modification of tyrosine residues. 2. Modification of 1 tyrosine residue/subunit does not affect the enzymic activity but decreases the response of the enzyme to the allosteric inhibitor, GTP. 3. The physical properties of the enzyme (sedimentation coefficient and optical rotatory dispersion) remain unaltered. 4. GTP partially protects against desensitization. 5. The diminished responses of the modified enzymes to GTP are also detected by using the fluorescence of 1-anilinonaphthalene-8-sulphonate as a conformational probe. 6. Difficulties that generally arise in chemical modifications from inhomogeneous distributions of products are discussed.

scholarly journals Chemical modification of amino groups and guanidino groups of trypsin. Preparation of stable and soluble derivatives

1975 ◽  
Vol 147 (1) ◽  
pp. 71-81 ◽  
Author(s):  
A Nureddin ◽  
T Inagami
Keyword(s):  
Chemical Modification ◽  
Peptide Bond ◽  
Enzymic Activity ◽  
Native Enzyme ◽  
Amino Group ◽  
Amino Groups ◽  
Appreciable Change ◽  
Neutral Ph ◽  
Arginine Residues ◽  
Esterolytic Activity

1. Isoionic chemical modification of amino groups of trypsin (EC 3.4.21.4) was studied for the purpose of obtaining a well-defined modified trypsin with minimum changes in physicochemical properties and with sufficient stability at neutral pH. Acetamidination with methyl acetimidate hydrochloride proceeded very rapidly at pH9.8 and 5degrees C and all 14 epsilon-amino groups were modified in 2h. The reaction was limited to epsilon-amino groups. The α-amino group of N-terminal isoleucine was modified only by repeated reactions in the presence of 5.5 M-guanidine or 8 M-urea. 2. The epsilon-acetamidinated derivative of β-trypsin retained enzymic activity at values comparable with those of native enzyme tested with α-N-benzoyl-L-arginine ethyl ester and α-N-benzoyl-L-arginine p-nitroanilide as substrates; it also showed substrate activation comparable with that of native enzyme. The acetamidination of α-trypsin resulted in approx. 50% decrease in its esterolytic activity. 3. The epsilon-acetamidinated β-trypsin was very stable at pH8 and 25degrees C in the absence of Ca2+. The activity of 0.04% (W/V) enzyme solution remained practically unchanged for 10h, and after 24h 90% of the activity was still retained. Possible autolytic cleavage of peptide bonds of acetamidinated enzymes was followed by N-terminal analysis by using automated Edman degradation. Only the Arg(105)-Val(106) bond was found to be cleaved to an appreciable extent. Thus β-trypsin can be stabilized simply by complete acetamidination of epsilon-amino groups without modifying guanidino groups of arginine residues. Acetamidinated α-trypsin was unstable, but its inactivation at a neutral pH could not be attributed to the cleavage of a single specific peptide bond. 4. The acetamidination of the α-amino group of the N-terminal isoleucine results in the inactivation of esterolytic activity. However, this enzyme retained the ability to react with p-nitrophenyl p'-guanidinobenzoate. 5. It was concluded that acetamidination of β-trypsin is a convenient method for preparing a well-defined stable and soluble trypsin derivative without appreciable change in its physical properties.

scholarly journals The reversible reaction of protein amino groups with exo-cis-3,6-endoxo-Δ4-tetrahydrophthalic anhydride. The reaction with lysozyme

1970 ◽  
Vol 118 (5) ◽  
pp. 733-739 ◽  
Author(s):  
M. Riley ◽  
R. N. Perham
Keyword(s):  
Free Amino ◽  
Tertiary Structure ◽  
Complete Recovery ◽  
Enzymic Activity ◽  
Carboxyl Group ◽  
Amino Group ◽  
Half Time ◽  
Amino Groups ◽  
Model Compounds ◽  
Anchimeric Assistance

1. The reaction of exo-cis-3,6-endoxo-Δ4-tetrahydrophthalic anhydride with amino groups of model compounds and lysozyme is described. 2. Reaction with the ∈-amino group of Nα-acetyl-l-lysine amide gives rise to two diastereoisomeric products; at acid pH the free amino group is liberated with anchimeric assistance by the neighbouring protonated carboxyl group with a half-time of 4–5h at pH3.0 and 25°C. 3. The amino groups of lysozyme can be completely blocked, with total loss of enzymic activity. Dialysis at pH3.0 results in complete recovery of the native primary and tertiary structure of lysozyme and complete return of catalytic activity. 4. The specificity of reaction of this and other anhydrides with amino groups in proteins is discussed.

scholarly journals The reaction of 2,4,6-trinitrobenzenesulphonic acid with amino acids, peptides and proteins

1968 ◽  
Vol 108 (3) ◽  
pp. 383-391 ◽  
Author(s):  
R. B. Freedman ◽  
G. K. Radda
Keyword(s):  
Amino Acids ◽  
Glutamate Dehydrogenase ◽  
Free Amino ◽  
Reactive Species ◽  
Second Order ◽  
Amino Group ◽  
Exponential Rate ◽  
Amino Groups ◽  
Sh Group ◽  
Kinetics Of

1. The kinetics of the reaction of 2,4,6-trinitrobenzenesulphonic acid with various amino acids, peptides and proteins were studied by spectrophotometry. 2. The reaction of the α- and ∈-amino groups in simple amino acids was found to be second-order, and the unprotonated amino group was shown to be the reactive species. 3. By allowing for the concentration of unreactive −NH3+ group, intrinsic reactivities for the free amino groups were derived and shown to be correlated with the basicities. 4. The SH group of N-acetylcysteine was found to be more reactive to 2,4,6-trinitrobenzenesulphonic acid than most amino groups. 5. The reactions of insulin, chymotrypsinogen and ribonuclease with 2,4,6-trinitrobenzenesulphonic acid were analysed in terms of three exponential rate curves, each referring to one or more amino groups of the proteins. 6. The reaction of lysozyme with 2,4,6-trinitrobenzenesulphonic acid was found to display an acceleration effect. 7. From the reaction of 2,4,6-trinitrobenzenesulphonic acid with glutamate dehydrogenase at several enzyme concentrations, it was possible to discern two sets of amino groups of different reactivity, and to show that the number of groups in each set was decreased by aggregation of the enzyme.

scholarly journals Homoserine and diaminobutyric acid in the mucopeptide-precursor-nucleotides and cell walls of some plant-pathogenic corynebacteria

1971 ◽  
Vol 121 (3) ◽  
pp. 417-423 ◽  
Author(s):  
H. R. Perkins
Keyword(s):  
Cell Wall ◽  
Cell Walls ◽  
Optical Rotatory Dispersion ◽  
Rotatory Dispersion ◽  
Cross Linking ◽  
Amino Group ◽  
Optical Rotatory ◽  
Corynebacterium Sepedonicum ◽  
Diaminobutyric Acid ◽  
The Third

The nucleotide precursors of cell-wall mucopeptide were prepared by vancomycin inhibition of Corynebacterium insidiosum and Corynebacterium poinsettiae. The amino acid in the third position in the former peptide was shown by optical rotatory dispersion of the bisdinitrophenyl derivative to be l-diaminobutyric acid. Homoserine in the nucleotide from C. poinsettiae was catalytically oxidized to aspartic acid, which was then shown to be the l-isomer by optical rotatory dispersion of the trinitrophenyl compound. The sequence for C. insidiosum was UDP-N-acetylmuramyl-glycyl-isoglutamyl- (γ-acetyl)-diaminobutyryl-alanyl-alanine. The cell walls of C. insidiosum and Corynebacterium sepedonicum contained diaminobutyric acid that was optically inactive. It is proposed, therefore, that the primary peptide chain contains the l-isomer with its γ-amino group blocked by acetylation and that cross-linking is achieved by means of the d-isomer, analogous to d-ornithine in C. poinsettiae.

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