Identification of two cysteine residues forming a pair of vicinal thiols in glucosamine-6-phosphate deaminase from Escherichia coli and a study of their functional role by site-directed mutagenesis

Biochemistry ◽  
1992 ◽  
Vol 31 (4) ◽  
pp. 1153-1158 ◽  
Author(s):  
Myriam M. Altamirano ◽  
Jacqueline A. Plumbridge ◽  
Mario L. Calcagno
Keyword(s):  
Escherichia Coli ◽  
Functional Role ◽  
Site Directed Mutagenesis ◽  
Cysteine Residues ◽  
Directed Mutagenesis ◽  
Vicinal Thiols

scholarly journals The role of cysteine residues 129 and 329 in Escherichia coli K1 CMP-NeuAc synthase

1993 ◽  
Vol 295 (2) ◽  
pp. 485-491 ◽  
Author(s):  
G Zapata ◽  
P P Roller ◽  
J Crowley ◽  
W F Vann
Keyword(s):  
Escherichia Coli ◽  
Specific Activity ◽  
Site Directed Mutagenesis ◽  
Cysteine Residues ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Acetylneuraminic Acid ◽  
Denatured States ◽  
Escherichia Coli K1

N-Acetylneuraminic acid cytidyltransferase (CMP-NeuAc synthase) of Escherichia coli K1 is sensitive to mercurials and has cysteine residues only at positions 129 and 329. The role of these residues in the catalytic activity and structure of the protein has been investigated by site-directed mutagenesis and chemical modification. The enzyme is inactivated by the thiol-specific reagent dithiodipyridine. Inactivation by this reagent is decreased in the presence of the nucleotide substrate CTP, suggesting that a thiol residue is at or near the active site. Site-directed mutagenesis of either residue Cys-129 to serine or Cys-329 to selected amino acids has minor effects on the specific activity of the enzyme, suggesting that cysteine is not essential for catalysis and that a disulphide bond is not an essential structural component. The limited reactivity of the enzyme to other thiol-blocking reagents suggests that its cysteine residues are partially exposed. The accessibility and role of the cysteine residues in enzyme structure were investigated by fluorescence, c.d. and denaturation studies of wild-type and mutant enzymes. The mutation of Cys-129 to serine makes the enzyme more sensitive to heat and chemical denaturation, but does not cause gross changes in the protein structure as judged by the c.d. spectrum. The mutant containing Ser-129 instead of Cys-129 had a complex denaturation pathway similar to that of wild-type E. coli K1 CMP-NeuAc synthase consisting of several partially denatured states. Cys-329 reacts more readily with N-[14C]ethylmaleimide when the enzyme is in a heat-induced relaxed state. Cys-129 is less reactive and is probably a buried residue.

Site-directed mutagenesis of cysteine residues in Escherichia coli fumarate reductase.

1991 ◽  
Vol 43 (2-3) ◽  
pp. 270 ◽  
Author(s):  
Mark T. Werth ◽  
Gary Cecchini ◽  
Annamaria Manodori ◽  
Brian A.C. Ackrell ◽  
Imke Schröder ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Fumarate Reductase ◽  
Site Directed Mutagenesis ◽  
Cysteine Residues ◽  
Directed Mutagenesis

Investigation of the functional role of tryptophan-22 in Escherichia coli dihydrofolate reductase by site-directed mutagenesis

Biochemistry ◽  
1991 ◽  
Vol 30 (46) ◽  
pp. 11092-11103 ◽  
Author(s):  
Mark S. Warren ◽  
Katherine A. Brown ◽  
Martin F. Farnum ◽  
Elizabeth E. Howell ◽  
Joseph Kraut
Keyword(s):  
Escherichia Coli ◽  
Dihydrofolate Reductase ◽  
Functional Role ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis

Probing the functional role of phenylalanine-31 of Escherichia coli dihydrofolate reductase by site-directed mutagenesis

Biochemistry ◽  
1987 ◽  
Vol 26 (13) ◽  
pp. 4093-4100 ◽  
Author(s):  
Jin Tann Chen ◽  
Kazunari Taira ◽  
Chen Pei D. Tu ◽  
Stephen J. Benkovic
Keyword(s):  
Escherichia Coli ◽  
Dihydrofolate Reductase ◽  
Functional Role ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis

scholarly journals Identification of an essential cysteine residue in human glutathione synthase

1997 ◽  
Vol 321 (1) ◽  
pp. 207-210 ◽  
Author(s):  
Rayappa R. GALI ◽  
Philip G. BOARD
Keyword(s):  
Functional Role ◽  
Residual Activity ◽  
Cysteine Residue ◽  
Enzymic Activity ◽  
Significant Inhibition ◽  
Site Directed Mutagenesis ◽  
Cysteine Residues ◽  
Directed Mutagenesis ◽  
Cellular Functions

Glutathione is essential for a variety of cellular functions, and is synthesized from γ-glutamylcysteine and glycine by the action of glutathione synthase (EC 6.3.2.3). Human glutathione synthase is a dimer of two identical subunits, each composed of 474 amino acids. Little is known about the structure–function relationships of mammalian glutathione synthases and, in order to gain a greater understanding of this critical enzyme, we have probed the role of cysteine residues by chemical modification and site-directed mutagenesis. Preincubation with thiol reagents such as p-chloromercuribenzoate, N-ethylmaleimide, iodoacetate and 5,5´-dithiobis-(2-nitrobenzoate) resulted in significant inhibition of recombinant human glutathione synthase. Each subunit contains cysteine residues at positions 294, 409 and 422, and we have prepared four different mutants by replacing individual cysteine residues, or all of the cysteine residues, with alanine. The C294A and C409A cysteine mutants retained significant residual activity, indicating that these two cysteine residues are not essential for activity. In contrast, substantial decreases in enzymic activity were detected with the C422A and cysteine-free mutants. This suggests that Cys-422 may play a significant structural or functional role in human glutathione synthase.

scholarly journals Role of cysteine residues in ribonuclease H from Escherichia coli. Site-directed mutagenesis and chemical modification

1990 ◽  
Vol 271 (1) ◽  
pp. 59-66 ◽  
Author(s):  
S Kanaya ◽  
S Kimura ◽  
C Katsuda ◽  
M Ikehara
Keyword(s):  
Escherichia Coli ◽  
Chemical Modification ◽  
Steric Hindrance ◽  
Thiol Group ◽  
Site Directed Mutagenesis ◽  
Ribonuclease H ◽  
Cysteine Residues ◽  
Directed Mutagenesis ◽  
E Coli

The role of the three cysteine residues at positions 13, 63 and 133 in Escherichia coli RNAase H, an enzyme that is sensitive to N-ethylmaleimide [Berkower, Leis & Hurwitz (1973) J. Biol. Chem. 248, 5914-5921], was examined by using both site-directed mutagenesis and chemical modification. Novel aspects that were found are as follows. First, none of the cysteine residues is required for activity. Secondly, chemical modification of either Cys-13 or Cys-133 with thiol-blocking reagents inactivates the enzyme, but that of Cys-63 does not. Thus the sensitivity of E. coli RNAase H to N-ethylmaleimide arises not from blocking of the thiol group but from steric hindrance by the modifying group incorporated at either Cys-13 or Cys-133.

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