TITRATION CALORIMETRIC ANALYSIS OF ACYLCOA RECOGNITION BY MYRISTOYLCOA-PROTEIN N-MYRISTOYLTRANSFERASE

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 displa y remarkable in vivo specificity for this rare acyl chain. To better u nderstand the mechanisms underlying this specificity, we have used iso thermal titration calorimetry as well as kinetic measurements to study the interactions of Nmt1p with acylCoA analogs having variations in c hain length and/or conformation, analogs with alterations in the thioe ster bond, and analogs with or without a 3'-phosphate in their CoA moi ety. MyristoylCoA binds to Nmt1p with a K-d of 15 nM and a large exoth ermic Delta H (-25 kcal/mol). CoA derivatives of C12:0-C16:0 fatty aci ds bind to Nmt1p with similar affinity, but with much smaller Delta H and a correspondingly less negative T Delta S than myristoylCoA. Repla cing the thioester carbonyl group with a methylene or removing the 3'- phosphate of CoA is each sufficient to prevent the low enthalpy bindin g observed with myristoylCoA. The carbonyl and the 3'-phosphate have d istinct and important roles in chain length recognition over the range C12-C16. Acyltransferase activity parallels binding enthalpy, The nat urally occurring cis-5-tetradecenoylCoA and cis-5,8-tetradecadienoylCo A are used as alternative Nmt substrates in retinal photoreceptor cell s, even though they do not exhibit in vitro kinetic or thermodynamic p roperties that are superior to those of myristoylCoA. The binding of a n 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 t hat full understanding of how protein acylation is controlled not only requires consideration of the acyltransferase and its peptide substra tes but also consideration of the synthesis and/or presentation of its lipid substrates.