Binding Affinity Determines Substrate Specificity and Enables Discovery of Substrates for N‑Myristoyltransferases

Kinetic parameters
(kcat and Km) derived from the Michaelis–Menten
equation are widely used to characterize enzymes. kcat/Km is considered the catalytic
efficiency or substrate specificity of an enzyme toward its substrate.
N-Myristoyltransferases (NMTs) catalyze the N-terminal glycine myristoylation
of numerous eukaryotic proteins. Surprisingly, we find that in vitro
human NMT1 can accept acetyl-CoA and catalyze acetylation with kcat and Km values
similar to that of myristoylation. However, when both acetyl-CoA and
myristoyl-CoA are present in the reaction, NMT1 catalyzes almost exclusively
myristoylation. This phenomenon is caused by the dramatically different
binding affinities of NMT1 for myristoyl-CoA and acetyl-CoA (estimated Kd of 14.7 nM and 10.1 μM, respectively).
When both are present, NMT1 is essentially entirely bound by myristoyl-CoA
and thus catalyzes myristoylation exclusively. The NMT1 example highlights
the crucial role of binding affinity in determining the substrate
specificity of enzymes, which in contrast to the traditionally held
view in enzymology that the substrate specificity is defined by kcat/Km values. This
understanding readily explains the vast biological literature showing
the coimmunoprecipitation of enzyme–substrate pairs for enzymes
that catalyzes protein post-translational modifications (PTM), including
phosphorylation, acetylation, and ubiquitination. Furthermore, this
understanding allows the discovery of substrate proteins by identifying
the interacting proteins of PTM enzymes, which we demonstrate by identifying
three previously unknown substrate proteins (LRATD1, LRATD2, and ERICH5)
of human NMT1/2 by mining available interactome data.