scholarly journals Cysteine residues in the Na+/dicarboxylate co-transporter, NaDC-1

1999 ◽  
Vol 344 (1) ◽  
pp. 205-209 ◽  
Author(s):  
Ana M. PAJOR ◽  
Sally J. KRAJEWSKI ◽  
Nina SUN ◽  
Rama GANGULA
Keyword(s):  
Protein Trafficking ◽  
Direct Relationship ◽  
Transmembrane Domain ◽  
Cysteine Residue ◽  
Site Directed Mutagenesis ◽  
Cysteine Residues ◽  
Directed Mutagenesis ◽  
Transport Activity ◽  
Specific Reagent

The role of cysteine residues in the Na+/dicarboxylate co-transporter (NaDC-1) was tested using site-directed mutagenesis. The transport activity of NaDC-1 was not affected by mutagenesis of any of the 11 cysteine residues, indicating that no individual cysteine residue is necessary for function. NaDC-1 is sensitive to inhibition by the impermeant cysteine-specific reagent, p-chloromercuribenzenesulphonate (pCMBS). The pCMBS-sensitive residues in NaDC-1 are Cys-227, found in transmembrane domain 5, and Cys-476, located in transmembrane domain 9. Although cysteine residues are not required for function in NaDC-1, their presence appears to be important for protein stability or trafficking to the plasma membrane. There was a direct relationship between the number of cysteine residues, regardless of location, and the transport activity and expression of NaDC-1. The results indicate that mutagenesis of multiple cysteine residues in NaDC-1 may alter the shape or configuration of the protein, leading to alterations in protein trafficking or stability.

scholarly journals Cysteine 144 in the Third Transmembrane Domain of the Creatine Transporter Is Located Close to a Substrate-binding Site

2001 ◽  
Vol 276 (50) ◽  
pp. 46983-46988 ◽  
Author(s):  
Joanna R. Dodd ◽  
David L. Christie
Keyword(s):  
Binding Site ◽  
Transmembrane Domain ◽  
Cysteine Residue ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Transport Activity ◽  
Creatine Transporter ◽  
The Third ◽  
Low Concentrations ◽  
Rapid Inactivation

All creatine transporters contain a cysteine residue (Cys144) in the third transmembrane domain that is not present in other members of the Na+,Cl−-dependent family of neurotransmitter transporters. Site-directed mutagenesis and reaction with methane thiosulfonates were used to investigate the importance of Cys144for transporter function. Replacement of Cys144with Ser did not significantly affect the kinetics or activity of the transporter, whereas a C144A mutant had a higherKm(0.33 compared with 0.18 mm). Substitution of Cys144with Leu gave a mutant with a 5-fold higherKmand a reduced specificity for substrate. Low concentrations of 2-aminoethyl methanethiosulfonate (MTSEA) resulted in rapid inactivation of the creatine transporter. The C144S mutant was resistant to inactivation, indicating that modification of Cys144was responsible for the loss of transport activity. Creatine and analogues that function as substrates of the creatine transporter were able to protect from MTSEA inactivation. Na+and Cl−ions were not necessary for MTSEA inactivation, but Na+was found to be important for creatine protection from inactivation. Our results indicate that cysteine 144 is close to the binding site or part of a permeation channel for creatine.

scholarly journals Cysteine residues in the organic anion transporter mOAT1

2004 ◽  
Vol 380 (1) ◽  
pp. 283-287 ◽  
Author(s):  
Kunihiko TANAKA ◽  
Fanfan ZHOU ◽  
Kogo KUZE ◽  
Guofeng YOU
Keyword(s):  
Organic Anion ◽  
Cysteine Residue ◽  
The Body ◽  
Organic Anion Transporter ◽  
Site Directed Mutagenesis ◽  
Cysteine Residues ◽  
Transport Activity ◽  
Anion Transporters ◽  
Anion Transporter

Mouse organic anion transporter 1 (mOAT1) belongs to a family of organic anion transporters, which play critical roles in the body disposition of clinically important drugs, including anti-HIV therapeutics, anti-tumour drugs, antibiotics, anti-hypertensives and anti-inflammatories. mOAT1-mediated transport of organic anion PAH (p-aminohippurate) in HeLa cells was inhibited by the cysteine-modifying reagent PCMBS (p-chloromercuribenzenesulphonate). Therefore the role of cysteine residues in the function of mOAT1 was examined by site-directed mutagenesis. All 13 cysteine residues in mOAT1 were replaced by alanine, singly or in combination. Single replacement of these residues had no significant effect on mOAT1-mediated PAH transport, indicating that no individual cysteine residue is necessary for function. Multiple replacements at a C-terminal region (C335/379/427/434A; Cys335/379/427/434→Ala) resulted in a substantial decrease in transport activity. A simultaneous replacement of all 13 cysteine residues (C-less) led to a complete loss of transport function. The decreased or lack of transport activity of the mutants C335/379/427/434A and C-less was due to the impaired trafficking of the mutant transporters to the cell surface. These results suggest that although cysteine residues are not required for function in mOAT1, their presence appears to be important for the targeting of the transporter to the plasma membrane. We also showed that, although all cysteine mutants of mOAT1 were sensitive to the inhibition by PCMBS, C49A was less sensitive than the wild-type mOAT1, suggesting that the modification of Cys49 may play a role in the inhibition of mOAT1 by PCMBS.

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 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.

The role of cysteine residues of spinach ferredoxin-NADP+ reductase as assessed by site-directed mutagenesis

Biochemistry ◽  
1993 ◽  
Vol 32 (25) ◽  
pp. 6374-6380 ◽  
Author(s):  
Alessandro Aliverti ◽  
Luciano Piubelli ◽  
Giuliana Zanetti ◽  
Thomas Luebberstedt ◽  
Reinhold G. Herrmann ◽  
...  
Keyword(s):  
Site Directed Mutagenesis ◽  
Cysteine Residues ◽  
Directed Mutagenesis

scholarly journals Probing the Role of Cysteine Residues in Glucosamine-1-Phosphate Acetyltransferase Activity of the Bifunctional GlmU Protein fromEscherichia coli: Site-Directed Mutagenesis and Characterization of the Mutant Enzymes

1998 ◽  
Vol 180 (18) ◽  
pp. 4799-4803 ◽  
Author(s):  
Frédérique Pompeo ◽  
Jean van Heijenoort ◽  
Dominique Mengin-Lecreulx
Keyword(s):  
Active Site ◽  
Cysteine Residue ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Acetyl Coa ◽  
Mutant Proteins ◽  
Acetyltransferase Activity ◽  
Phosphate Acetyltransferase ◽  
High Level

ABSTRACT The glucosamine-1-phosphate acetyltransferase activity but not the uridyltransferase activity of the bifunctional GlmU enzyme fromEscherichia coli was lost when GlmU was stored in the absence of β-mercaptoethanol or incubated with thiol-specific reagents. The enzyme was protected from inactivation in the presence of its substrate acetyl coenzyme A (acetyl-CoA), suggesting the presence of an essential cysteine residue in or near the active site of the acetyltransferase domain. To ascertain the role of cysteines in the structure and function of the enzyme, site-directed mutagenesis was performed to change each of the four cysteines to alanine, and plasmids were constructed for high-level overproduction and one-step purification of histidine-tagged proteins. Whereas the kinetic parameters of the bifunctional enzyme appeared unaffected by the C296A and C385A mutations, 1,350- and 8-fold decreases of acetyltransferase activity resulted from the C307A and C324A mutations, respectively. TheKm values for acetyl-CoA and GlcN-1-P of mutant proteins were not modified, suggesting that none of the cysteines was involved in substrate binding. The uridyltransferase activities of wild-type and mutant GlmU proteins were similar. From these studies, the two cysteines Cys307 and Cys324 appeared important for acetyltransferase activity and seemed to be located in or near the active site.

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