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 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.

Role of lysines in human angiogenin: chemical modification and site-directed mutagenesis

Biochemistry ◽  
1989 ◽  
Vol 28 (4) ◽  
pp. 1726-1732 ◽  
Author(s):  
Robert Shapiro ◽  
Edward A. Fox ◽  
James F. Riordan
Keyword(s):  
Chemical Modification ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis

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.

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