scholarly journals Determination of the Nucleotide Binding Site withinClostridium symbiosumPyruvate Phosphate Dikinase by Photoaffinity Labeling, Site-Directed Mutagenesis, and Structural Analysis†

Biochemistry ◽  
1996 ◽  
Vol 35 (26) ◽  
pp. 8544-8552 ◽  
Author(s):  
Marielena McGuire ◽  
Lawrence J. Carroll ◽  
Linda Yankie ◽  
Sara H. Thrall ◽  
Debra Dunaway-Mariano ◽  
...  
Keyword(s):  
Structural Analysis ◽  
Binding Site ◽  
Photoaffinity Labeling ◽  
Nucleotide Binding ◽  
Nucleotide Binding Site ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis

Photoaffinity labeling of the nucleotide binding site of actin

Biochemistry ◽  
1986 ◽  
Vol 25 (19) ◽  
pp. 5793-5798 ◽  
Author(s):  
G. Hegyi ◽  
L. Szilagyi ◽  
M. Elzinga
Keyword(s):  
Binding Site ◽  
Photoaffinity Labeling ◽  
Nucleotide Binding ◽  
Nucleotide Binding Site

scholarly journals Definition of a Nucleotide Binding Site on Cytochromecby Photoaffinity Labeling

1996 ◽  
Vol 271 (31) ◽  
pp. 18379-18386 ◽  
Author(s):  
David B. McIntosh ◽  
Jonathan C. Parrish ◽  
Carmichael J. A. Wallace
Keyword(s):  
Binding Site ◽  
Photoaffinity Labeling ◽  
Nucleotide Binding ◽  
Nucleotide Binding Site ◽  
Definition Of

scholarly journals The Nucleotide-binding Site of Human Sphingosine Kinase 1

2002 ◽  
Vol 277 (51) ◽  
pp. 49545-49553 ◽  
Author(s):  
Stuart M. Pitson ◽  
Paul A. B. Moretti ◽  
Julia R. Zebol ◽  
Reza Zareie ◽  
Claudia K. Derian ◽  
...  
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Sphingosine Kinase ◽  
Structural Similarity ◽  
Nucleotide Binding ◽  
Nucleotide Binding Site ◽  
Site Directed Mutagenesis ◽  
Sequence Motifs ◽  
Sphingosine Kinase 1 ◽  
Nucleotide Binding Sites

Sphingosine kinase catalyzes the formation of sphingosine 1-phosphate, a lipid second messenger that has been implicated in a number of agonist-driven cellular responses including mitogenesis, anti-apoptosis, and expression of inflammatory molecules. Despite the importance of sphingosine kinase, very little is known regarding its structure or mechanism of catalysis. Moreover, sphingosine kinase does not contain recognizable catalytic or substrate-binding sites, based on sequence motifs found in other kinases. Here we have elucidated the nucleotide-binding site of human sphingosine kinase 1 (hSK1) through a combination of site-directed mutagenesis and affinity labeling with the ATP analogue, FSBA. We have shown that Gly82of hSK1 is involved in ATP binding since mutation of this residue to alanine resulted in an enzyme with an ∼45-fold higherKm(ATP). We have also shown that Lys103is important in catalysis since an alanine substitution of this residue ablates catalytic activity. Furthermore, we have shown that this residue is covalently modified by FSBA. Our data, combined with amino acid sequence comparison, suggest a motif of SGDGX17–21K is involved in nucleotide binding in the sphingosine kinases. This motif differs in primary sequence from all previously identified nucleotide-binding sites. It does, however, share some sequence and likely structural similarity with the highly conserved glycine-rich loop, which is known to be involved in anchoring and positioning the nucleotide in the catalytic site of many protein kinases.

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