Pre-steady-state kinetic studies of Saccharomyces cerevisiae myristoylCoA:protein N-myristoyltransferase mutants identify residues involved in catalysis.
MyristoylCoA:protein N-myristoyltransferase (Nmt, EC 2.3.1.97), a member of the GCN5 acetyltransferase (GNAT) superfamily, is an essential eukaryotic enzyme that catalyzes covalent attachment of myristate (C14:0) to the N-terminal Gly of proteins involved in myriad cellular functions. The 2.5 A resolution structure of a ternary complex of Saccharomyces cerevisiae Nmt1p with a bound substrate peptide (GLYASKLA) and nonhydrolyzable myristoylCoA analogue [Farazi, T. A., et al. (2001) Biochemistry 40, 6335] was used as the basis for a series of mutagenesis experiments designed to define the enzyme's catalytic mechanism. The kinetic properties of an F170A/L171A Nmt mutant are consistent with the proposal that their main chain amides, located in a beta-bulge structure conserved among GNATs, function as an oxyanion hole to polarize the thioester carbonyl of bound myristoylCoA prior to subsequent nucleophilic attack. Removal of the two C-terminal residues (M454 and L455) produces a 300--400-fold reduction in the chemical transformation rate and converts the rate-limiting step from a step after the transformation to the transformation event itself. This finding is consistent with the main chain C-terminal carboxylate of L455 functioning as a catalytic base that abstracts a proton from the N-terminal Gly ammonium of the bound peptide to generate the nucleophilic amine. Mutating N169 and T205 in concert reduces the rate of the chemical transformation, supporting their role as components of an H-bonding network that facilitates attack of the Gly1 amine and stabilizes the tetrahedral intermediate.
Farazi
T A
TA
Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Manchester
J K
JK
Waksman
G
G
Gordon
J I
JI
D000217:Acyltransferases / Q000235:genetics* / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism; D000409:Alanine / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics*; D000818:Animals; D053502:Arsenate Reductases; D053501:Arsenite Transporting ATPases; D001216:Asparagine / Q000235:genetics; D002384:Catalysis; D002417:Cattle; D016623:Ion Pumps; D007700:Kinetics; D007930:Leucine / Q000235:genetics; D009097:Multienzyme Complexes; D016297:Mutagenesis, Site-Directed; D019814:Myristic Acid / Q000378:metabolism; D010446:Peptide Fragments / Q000235:genetics; D010649:Phenylalanine / Q000235:genetics; D011817:Rabbits; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D017384:Sequence Deletion; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity / Q000235:genetics; D013552:Swine; D013912:Threonine / Q000235:genetics
D016623:Ion Pumps; D009097:Multienzyme Complexes; D010446:Peptide Fragments; D029701:Saccharomyces cerevisiae Proteins; D019814:Myristic Acid; D013912:Threonine; D010649:Phenylalanine; D001216:Asparagine; C506971:ARR2 protein, S cerevisiae; D053502:Arsenate Reductases; D000217:Acyltransferases; C050480:glycylpeptide N-tetradecanoyltransferase; D000251:Adenosine Triphosphatases; D053501:Arsenite Transporting ATPases; D007930:Leucine; D000409:Alanine