Effects of l-Histidine and Its Structural Analogues on Human N-Myristoyltransferase Activity and Importance of EEVEH Amino Acid Sequence for Enzyme Activity

Abstract

Myristoyl-CoA:protein N-myristoyltransferase (NMT) is an essential eukaryotic enzyme that catalyzes the cotranslational transfer of myristate to the NH2-terminal glycine residue of a number of important proteins of diverse function. Human NMT (hNMT) activity was found to be activated by L-histidine in a concentration-dependent manner. In contrast, two structural analogues of L-histidine, L-histidinol and histamine, inhibited hNMT activity in a noncompetitive manner with half-maximal inhibitions of 18 and 1.5 mM, respectively. The inhibition of hNMT activity by L-histidinol was reversed by a 2-fold molar excess of L-histidine, suggesting that L-histidine and L-histidinol were competing for a common site on NMT. Kinetic data indicated that whereas L-histidine enhanced the Vmax, both L-histidinol and histamine decreased the Vmax; none of these compounds altered the Km. Our studies suggest that L-histidine and its analogues may be interacting with His-293, involved in myristoyl-CoA transfer, rather than His-218, and implicated in the transfer of myristoyl-CoA to the peptide substrates. Site-directed mutagenesis of His-293, Val-291, and Glu-290 resulted in proteins with no measurable NMT activity. The most conserved region in the catalytic domain EEVEH (289-293) is critical for the myristoyl-CoA transfer in the NMT-catalyzed reactions. This region will be useful for the design of regulators of NMT function.

N-Myristoyltransferase 1 Is Essential in Early Mouse Development

“…The production of the Nmt1-␤geo fusion protein was fortunate because it made it possible for us to assess Nmt1 expression in tissue sections with X-gal stains. The fusion protein would not be predicted to retain any NMT enzymatic activity, inasmuch as it would lack important functional domains of NMT, for example regions that bind the protein substrate and myristoyl-CoA (23,24).…”

Molecular Cloning, Genomic Organization, and Biochemical Characterization of Myristoyl-CoA:ProteinN-Myristoyltransferase from Arabidopsis thaliana

“…1). A high degree of sequence conservation was also found throughout these primary sequences, including the residues essential for myristoyl CoA binding, the peptide binding, and the active site of the enzyme (53)(54)(55). In addition, two signature sequences of NMT, "KFGXGDG" and "(ED)(IV)NFLXHK," are also conserved in AtNMT1 (Fig.…”

The Biology and Enzymology of ProteinN-Myristoylation

“…Different classes of Nmt inhibitors have been identified including analogs of myristate and myristoyl-CoA (30 -36), myristoylpeptide derivatives (2, 37), and histidine analogs (38). All studied Nmts exhibit a preference for myristoyl-CoA.…”