Scanning Alanine Mutagenesis and De-peptidization of aCandida albicansMyristoyl-CoA:ProteinN-Myristoyltransferase Octapeptide Substrate Reveals Three Elements Critical for Molecular Recognition

Charles A. McWherter, Warren J. Rocque, Mark E. Zupec, Sandra K. Freeman, David L. Brown, Balekudru Devadas, Daniel P. Getman, James A. Sikorski, Jeffrey I. Gordon
1997 Journal of Biological Chemistry  
Candida albicans produces a single myristoyl-CoA: protein N-myristoyltransferase (Nmt) that is essential for its viability. An ADP-ribosylation factor (Arf) is included among the few cellular protein substrates of this enzyme. An octapeptide (GLYASKLS-NH 2 ) derived from a N-terminal Arf sequence was used as the starting point to identify elements critical for recognition by the acyltransferases's peptide-binding site. In vitro kinetic studies, employing purified Nmt and a panel of peptides
more » ... single Ala substitutions at each position of GLYASKLS-NH 2 , established that its Gly 1 , Ser 5 , and Lys 6 residues play predominant roles in binding. ALYASKLS-NH 2 was found to be an inhibitor competitive for peptide (K i ‫؍‬ 15.3 ؎ 6.4 M) and noncompetitive for myristoyl-CoA (K i ‫؍‬ 31.2 ؎ 0.7 M). A survey of 26 derivatives of this inhibitor, representing (i) a complete alanine scan, (ii) progressive C-terminal truncations, and (iii) manipulation of the physical-chemical properties of its residues 1, 5, and 6, confirmed the important stereochemical requirements for the N-terminal amine, the ␤-hydroxyl of Ser 5 , and the ⑀-amino group of Lys 6 . Remarkably, replacement of the the N-terminal tetrapeptide of ALYASKLS-NH 2 with an 11-aminoundecanoyl group produced a competitive inhibitor, 11-aminoundecanoyl-SKLS-NH 2 , that was 38-fold more potent (K i ‫؍‬ 0.40 ؎ 0.03 M) than the starting octapeptide. Removing the primary amine (undecanoyl-SKLS-NH 2 ), or replacing it with a methyl group (dodecanoyl-SKLS-NH 2 ), resulted in 26-and 34fold increases in IC 50 , confirming the important contribution of the amine to recognition. Removal of Leu-Ser from the C terminus (11-aminoundecanoyl-SK-NH 2 ) yielded a competitive dipeptide inhibitor with a K i (11.7 ؎ 0.4 M) equivalent to that of the starting octapeptide, ALYASKLS-NH 2 . Substitution of Ser with homoserine, cis-4-hydroxyproline, or tyrosine reduces potency by 3-70-fold, emphasizing the requirement for proper presentation of the hydroxyl group in the dipeptide inhibitor. Substituting D-for L-Lys decreases its inhibitory activity >100-fold, while deletion of the ⑀-amino group (Nle) or masking its charge (⑀-N-acetyl-lysine) produces 4 -7-fold attenuations. L-His, but not its D-isomer, can fully substitute for L-Lys, producing a compet-itive dipeptide inhibitor with similar potency (K i ‫؍‬ 11.9 ؎ 1.0 M). 11-Aminoundecanoyl-SK-NH 2 and 11-aminoundecanoyl-SH-NH 2 establish that a simple alkyl backbone can maintain an appropriate distance between three elements critical for recognition by the fungal enzyme's peptide-binding site: a simple -terminal amino group, a ␤-hydroxyl, and an ⑀-amino group or an imidazole. These compounds contain one peptide bond and two chiral centers, suggesting that it may be feasible to incorporate these elements of recognition, or functionally equivalent mimics, into a fully de-peptidized Nmt inhibitor.
doi:10.1074/jbc.272.18.11874 pmid:9115247 fatcat:ctthf6oyfbb37cd3i2pkussvje