Problem 1 Protein N-myristoylation involves the covalent attachment of the fatty acid, myristate (CH3(CH)12CO), to the N-terminal glycine of a variety of cellular proteins. This type of reaction is one of many examples of a post-translational protein modification (or PTM). This particular reaction uses myristate-coenzyme A (myristate-SCOA) as a co-substrate and is catalyzed by an enzyme known as N-myristoyltransferase (NMT). H myristoyl-SCOA R +H3N NMT pH 7 12 N + HS H CoA-SH R + H+ (a) Propose two possible mechanisms for the N-myristoylation reaction. In one mechanism, assume there is an active site Cys and that the reaction occurs through a covalent intermediate. In the other, assume that a Glu residue is critical for enzyme catalysis. (b) Kinetics analysis of the myristoylation reaction catalyzed by NMT was performed by varying the concentration of myristoyl-CoA at fixed concentrations of a peptide substrate (panel A). The converse experiment in which peptide concentration is varied at fixed [myristoyl-CoA] was also conducted (panel B). From these data, estimate Kms for both myristoyl-CoA and the peptide substrate. (Note: assume that in both plots all lines converge to a single point on the x-axis.) A 1/v (1/pmol/min) -0.5 191 M peptide O 96.4 M peptide 47.7 M peptide 23.8 M peptide 0.5 1.0 1/[MyristoylCoA] (M-1) 0.0 1.5 B 1/v (1/pmol/min) 0 4.3 M Myr-CoA 228 M Myr-CoA 125 M Myr-CoA 0.74 M Myr-CoA -10 0 10 1/[GNAAAARR-NH) (M-1) x 103 20 30 40 50 (c) Given the data in part (b), can you distinguish between the two mechanisms in part (a). Briefly explain your reasoning.