Abstract
Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase (Nmt1p) is an essential, monomeric enzyme that catalyzes the transfer of myristate from CoA to the amino-terminal Gly residue of cellular proteins. Product inhibition studies indicate that Nmt1p has an ordered Bi Bi reaction mechanism with myristoyl-CoA binding to the apo-enzyme to form a high affinity binary complex followed by binding of peptide with subsequent release of CoA and then the myristoylpeptide product. We have used isothermal titration calorimetry to quantify the effects of varying acyl chain length and removing the 3'-phosphate group of CoA on the energetics of interaction between Nmt1p and acyl-CoA ligands. Myristoyl-CoA binds to apo-Nmt1p with an affinity of 15 nM, corresponding to a binding free energy of -10.9 kcal/mol. This free energy is composed of a large favorable enthalpy of -24 kcal/mol and a large unfavorable entropic term. This large negative delta H degrees is consistent with a conformational change in the enzyme upon ligation, allowing synthesis of a functional peptide binding site. Binding of palmitoyl-CoA and lauroyl-CoA is driven by an exothermic enthalpy change which is much smaller than the corresponding parameter for myristoyl-CoA binding. The large differences in binding enthalpy and entropy (delta delta H degrees and T delta delta S degrees = 8-9 kcal/mol) demonstrate that the "off-length" acyl-CoAs bind to Nmt1p in a significantly different energetic fashion from myristoyl-CoA, even though the enzyme does not have a great deal of specificity among these ligands in terms of binding free energy (delta delta G degrees < or = 1 kcal/mol). The effect of removing the CoA 3'-phosphate group from myristoyl-CoA is similar to the effect of a two-carbon change in acyl chain length: i.e. an enthalpy dominated reduction in binding affinity. However, kinetic studies reveal that removing the 3'-phosphate from myristoyl-CoA has little effect on Nmt1p's catalytic efficiency, indicating that the 3'-phosphate group contributes binding free energy but little catalytic destabilization. The greater delta delta G degrees, with smaller delta delta H degrees and delta delta S degrees components, produced by removing the 3'-phosphate compared to increasing chain length suggests that it is not primarily the interactions of the 3'-phosphate which are disrupted when palmitoyl-CoA is substituted for myristoyl-CoA. No detectable interactions were noted between apo-Nmt1p and the substrate peptide, GAAPSKIV-NH2, providing additional support for the preferred ordered reaction mechanism.(ABSTRACT TRUNCATED AT 400 WORDS)
Highlights
From the Department of Molecular Biology and Pharmacology, Fashington University School of Medicine, St
Nmtlp has an ordered Bi Bi reaction mechanism with nonhydrolyzable myristoyl-CoA or palmitoyl-CoA anamyristoyl-CoAbinding to theapo-enzymeto form a high logs.The 3.6 p~ dissociation constant for binding of affinity binary complex followed bybinding of peptide GAAPSKIV-NH, to Nmtlp~S-(2-oxo)pentadecyl-CoAis with subsequent release ofCoA and the myris- driven by favorable enthalpy (-6.5 kcal/mol) and entoylpeptide product
The fact that a high affinity calorimetry to quantify the effects of varying acyl chain peptide binding site is formed with a nonhydrolyzlength and removing the 3"phosphate group of CoA on able palmitoyl-CoA analogS,(2-oxo)heptadecyl-CoAi,nthe energetics of interaction between Nmtlp and acyl- dicates that theenergy of binding of the off-length acyl
Summary
Lauroyl-CoA [4] S-(2-Oxo)pentadecyl-CoA[7] S-(2-Oxo)heptadecyl-CoA[2] Myristoyl (3'-dephospho)-CoA( 5 ). Peptide + NmtlpS-(2-Oxo)alkyl-CoAternary complex formation GAAPSKIV-NH, peptide S-(2-Oxo)pentadecyl-CoA[2] GAAPSKIV-NH, peptide S-(2-Oxo)heptadecyl-CoA[2]. 0.92 f 0.01 0.94 f 0.003 eter was calibrated by generating a heat pulse in thereaction cell with This effect wasendothermic and constant over the concentration range a built-in calibration heater of known resistance. The reaction cell (1.35-mrleaction volume plus 0.75 ml of dead space) was subtracted from each integrated data point beforethe isotherm was was filledwith Nmtlp ata concentration appropriate to the reaction in fit. A stirrer-syringe containing a solution of ligand (typically100 times the Nmtlp concentration)was mounted on the apparatus and stimng at400 rpm was initiated. The cell was allowed to reach equilibrium (operationally defined by a stable base line).
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