Role of a cysteine residue in substrate entry and catalysis in MtHIBADH : analysis by chemical modifications and site‐directed mutagenesis

IUBMB Life ◽  
2021 ◽  
Author(s):  
Amrita Singh ◽  
Nagendar Goud Badepally ◽  
Avadhesha Surolia
Keyword(s):  
Cysteine Residue ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Chemical Modifications

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 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.

scholarly journals A conserved motif in the yeast nucleolar protein Nop2p contains an essential cysteine residue

1998 ◽  
Vol 337 (1) ◽  
pp. 29-35 ◽  
Author(s):  
Michelle KING ◽  
Duy TON ◽  
Kent L. REDMAN
Keyword(s):  
Ribosomal Subunit ◽  
Cysteine Residue ◽  
Nucleolar Protein ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Large Ribosomal Subunit ◽  
Conserved Residues ◽  
Conserved Motif ◽  
Yeast Viability

Nop2p is an essential nucleolar protein in Saccharomyces cerevisiae that is involved in large ribosomal subunit assembly. It has substantial homology with human p120, the proliferation-associated nucleolar antigen that is overexpressed in many human cancers. A motif containing an invariant Pro–Cys dipeptide is found in Nop2p, p120 and the bacterial Fmu proteins. A total of nine conserved residues, including Pro423 and Cys424, were individually altered in Nop2p by site-directed mutagenesis. Nop2p function was abolished by conversion of Cys424 into either alanine or serine. All of the other Nop2p mutations tested sustained yeast viability, including glycine replacement of Pro423 and the conversion of a second conserved cysteine into alanine. The crucial role of Cys424 in Nop2p is intriguing, due to the critical roles that cysteine residues adjacent to a proline have in a number of nucleotide-modifying enzymes.

Heparinase I from Flavobacterium heparinum: The Role of the Cysteine Residue in Catalysis as Probed by Chemical Modification and Site-Directed Mutagenesis

Biochemistry ◽  
1995 ◽  
Vol 34 (44) ◽  
pp. 14441-14448 ◽  
Author(s):  
Ram Sasisekharan ◽  
Deborah Leckband ◽  
Ranga Godavarti ◽  
Ganesh Venkataraman ◽  
Charles L. Cooney ◽  
...  
Keyword(s):  
Chemical Modification ◽  
Cysteine Residue ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Heparinase I ◽  
Flavobacterium Heparinum

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.

Role of Cysteine Residues in the N-Terminal Extracellular Domain of the Human VIP 1 Receptor for Ligand Binding A Site-directed Mutagenesis Studya

2006 ◽  
Vol 805 (1) ◽  
pp. 585-589 ◽  
Author(s):  
PASCALE GAUDIN ◽  
ALAIN COUVINEAU ◽  
JEAN-JOSÉ MAORET ◽  
CHRISTIANE ROUYER-FESSARD ◽  
MARC LABURTHE
Keyword(s):  
Ligand Binding ◽  
Extracellular Domain ◽  
Site Directed Mutagenesis ◽  
Cysteine Residues ◽  
Directed Mutagenesis ◽  
A Site

scholarly journals Site-directed mutagenesis of tyrosine hydroxylase. Role of serine 40 in catalysis.

1992 ◽  
Vol 267 (36) ◽  
pp. 25754-25758
Author(s):  
J Wu ◽  
D Filer ◽  
A.J. Friedhoff ◽  
M Goldstein
Keyword(s):  
Tyrosine Hydroxylase ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis
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