Titration Calorimetric Analysis of AcylCoA Recognition by MyristoylCoA:Protein N-Myristoyltransferase
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- 作者：Rajiv S. Bhatnagar ; Otto F. Schall ; Emily Jackson-Machelski ; James A. Sikorski ; Balekudru Devadas ; George W. Gokel ; and Jeffrey I. Gordon
- 刊名：Biochemistry
- 出版年：1997
- 出版时间：June 3, 1997
- 年：1997
- 卷：36
- 期：22
- 页码：6700 - 6708
- 全文大小：195K
- 年卷期：v.36,no.22(June 3, 1997)
- ISSN：1520-4995
文摘
Saccharomyces cerevisiae myristoylCoA:proteinN-myristoyltransferase (Nmt1p) is an essentialenzyme that catalyzes the transfer of myristic acid (C14:0) frommyristoylCoA to the N-terminus ofcellular proteins with a variety of functions. Nmts from anassortment of species display remarkable invivo specificity for this rare acyl chain. To betterunderstand the mechanisms underlying this specificity,we have used isothermal titration calorimetry as well as kineticmeasurements to study the interactions ofNmt1p with acylCoA analogs having variations in chain length and/orconformation, analogs with alterationsin the thioester bond, and analogs with or without a 3'-phosphate intheir CoA moiety. MyristoylCoAbinds to Nmt1p with a Kd of 15 nM and a largeexothermic 
H (-25 kcal/mol). CoA derivativesofC12:0-C16:0 fatty acids bind to Nmt1p with similar affinity, but withmuch smaller H and acorrespondingly less negative TS thanmyristoylCoA. Replacing the thioester carbonyl group withamethylene or removing the 3'-phosphate of CoA is each sufficient toprevent the low enthalpy bindingobserved with myristoylCoA. The carbonyl and the 3'-phosphate havedistinct and important roles inchain length recognition over the range C12-C16. Acyltransferaseactivity parallels binding enthalpy.The naturally occurring cis-5-tetradecenoylCoA andcis-5,8-tetradecadienoylCoA are used asalternativeNmt substrates in retinal photoreceptor cells, even though they do notexhibit in vitro kinetic orthermodynamic properties that are superior to those of myristoylCoA.The binding of an acylCoA is thefirst step in the enzyme's ordered reaction mechanism. Ourfindings suggest that within cells, limitationof Nmt substrate usage occurs through control of acylCoA availability.This indicates that full understandingof how protein acylation is controlled not only requires considerationof the acyltransferase and its peptidesubstrates but also consideration of the synthesis and/or presentationof its lipid substrates.
H (-25 kcal/mol). CoA derivativesofC12:0-C16:0 fatty acids bind to Nmt1p with similar affinity, but withmuch smaller H and acorrespondingly less negative TS thanmyristoylCoA. Replacing the thioester carbonyl group withamethylene or removing the 3'-phosphate of CoA is each sufficient toprevent the low enthalpy bindingobserved with myristoylCoA. The carbonyl and the 3'-phosphate havedistinct and important roles inchain length recognition over the range C12-C16. Acyltransferaseactivity parallels binding enthalpy.The naturally occurring cis-5-tetradecenoylCoA andcis-5,8-tetradecadienoylCoA are used asalternativeNmt substrates in retinal photoreceptor cells, even though they do notexhibit in vitro kinetic orthermodynamic properties that are superior to those of myristoylCoA.The binding of an acylCoA is thefirst step in the enzyme's ordered reaction mechanism. Ourfindings suggest that within cells, limitationof Nmt substrate usage occurs through control of acylCoA availability.This indicates that full understandingof how protein acylation is controlled not only requires considerationof the acyltransferase and its peptidesubstrates but also consideration of the synthesis and/or presentationof its lipid substrates.