HlyC, the Internal Protein Acyltransferase That Activates Hemolysin Toxin: The Role of Conserved Tyrosine and Arginine Residues in Enzymatic Activity As Probed by Chemical Modification and Site-Directed Mutagenesis
Internal fatty acylation of proteins is a recognized means of modifying biological behavior. Escherichia coli hemolysin A (HlyA), a toxic protein, is transcribed as a nontoxic protein and made toxic by internal acylation of two lysine residue ε-amino groups; HlyC catalyzes the acyl transfer from acy...
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Published in | Biochemistry (Easton) Vol. 38; no. 27; pp. 8831 - 8838 |
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Main Authors | , , |
Format | Journal Article |
Language | English |
Published |
United States
American Chemical Society
06.07.1999
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Subjects | |
Online Access | Get full text |
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Summary: | Internal fatty acylation of proteins is a recognized means of modifying biological behavior. Escherichia coli hemolysin A (HlyA), a toxic protein, is transcribed as a nontoxic protein and made toxic by internal acylation of two lysine residue ε-amino groups; HlyC catalyzes the acyl transfer from acyl−acyl carrier protein (ACP), the obligate acyl donor. Conserved residues among the respective homologous C proteins that activate 13 different RTX (repeats in toxin) toxins of which HlyA is the prototype likely include some residues that are important in catalysis. Possible roles of two conserved tyrosines and two conserved arginines were investigated by noting the effects of chemical modifiers and site-directed mutagenesis. TNM modification of HlyC at pH 8.0 led to extensive inhibition that was prevented by the presence of the substrate myristoyl-ACP but not by the product, ACPSH. NAI had no effect. Y70G and Y150G greatly diminished enzyme activity, whereas mutations Y70F and Y150F exhibited wild-type activity. Modification of arginine residues with PG markedly lowered acyltransferase activity with moderate protection by both myristoyl-ACP and ACPSH. Under optimum conditions, four separate mutations of the two conserved arginine residues (R24A, R24K, R87A, and R87K) had little effect on acyltransferase activity. |
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Bibliography: | istex:3ADFD52FEFC114015E733C4AB0817D8E05DDBB11 This work was supported in part by a grant-in-aid from the American Heart Association-Tennessee Affiliate, a grant from the American Heart Association, Southeast Affiliate, and National Institutes of Health Grant GM/OD54337. ark:/67375/TPS-SJ18F919-8 ObjectType-Article-2 SourceType-Scholarly Journals-1 ObjectType-Feature-1 content type line 23 |
ISSN: | 0006-2960 1520-4995 |
DOI: | 10.1021/bi990138y |