Titration Calorimetric Analysis of AcylCoA Recognition by MyristoylCoA:Protein N-Myristoyltransferase

• Published in: Biochemistry (Easton) Vol. 36; no. 22; pp. 6700 - 6708
• Main Authors: Bhatnagar, Rajiv S, Schall, Otto F, Jackson-Machelski, Emily, Sikorski, James A, Devadas, Balekudru, Gokel, George W, Gordon, Jeffrey I
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 03.06.1997
• Subjects: Acyl Coenzyme A - chemistry; Acyl Coenzyme A - metabolism; Acylation; Acyltransferases - chemistry; Acyltransferases - metabolism; Amino Acid Sequence; Calorimetry; Kinetics; Molecular Conformation; Phosphates - chemistry; Phosphates - metabolism; Recombinant Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae - enzymology; Structure-Activity Relationship; Substrate Specificity; Thermodynamics
• DOI: 10.1021/bi970311v
• ISSN: 0006-2960

Saccharomyces cerevisiae myristoylCoA:protein N-myristoyltransferase (Nmt1p) is an essential enzyme that catalyzes the transfer of myristic acid (C14:0) from myristoylCoA to the N-terminus of cellular proteins with a variety of functions. Nmts from an assortment of species display remarkable in vivo specificity for this rare acyl chain. To better understand the mechanisms underlying this specificity, we have used isothermal titration calorimetry as well as kinetic measurements to study the interactions of Nmt1p with acylCoA analogs having variations in chain length and/or conformation, analogs with alterations in the thioester bond, and analogs with or without a 3‘-phosphate in their CoA moiety. MyristoylCoA binds to Nmt1p with a K d of 15 nM and a large exothermic ΔH (−25 kcal/mol). CoA derivatives of C12:0−C16:0 fatty acids bind to Nmt1p with similar affinity, but with much smaller ΔH and a correspondingly less negative TΔS than myristoylCoA. Replacing the thioester carbonyl group with a methylene or removing the 3‘-phosphate of CoA is each sufficient to prevent the low enthalpy binding observed with myristoylCoA. The carbonyl and the 3‘-phosphate have distinct and important roles in chain length recognition over the range C12−C16. Acyltransferase activity parallels binding enthalpy. The naturally occurring cis-5-tetradecenoylCoA and cis-5,8-tetradecadienoylCoA are used as alternative Nmt substrates in retinal photoreceptor cells, even though they do not exhibit in vitro kinetic or thermodynamic properties that are superior to those of myristoylCoA. The binding of an acylCoA is the first step in the enzyme's ordered reaction mechanism. Our findings suggest that within cells, limitation of Nmt substrate usage occurs through control of acylCoA availability. This indicates that full understanding of how protein acylation is controlled not only requires consideration of the acyltransferase and its peptide substrates but also consideration of the synthesis and/or presentation of its lipid substrates.