Effects of L-histidine and its structural analogues on human N-myristoyltransferase activity and importance of EEVEH amino acid sequence for enzyme activity.
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.
Department of Pathology, Saskatoon Cancer Centre, College of Medicine, University of Saskatchewan, Canada.
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D000217:Acyltransferases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence / Q000235:genetics; D001665:Binding Sites; D002384:Catalysis; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D004789:Enzyme Activation / Q000235:genetics; D006639:Histidine / Q000031:analogs & derivatives* / Q000378:metabolism* / Q000494:pharmacology; D006641:Histidinol / Q000494:pharmacology; D006801:Humans; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D017384:Sequence Deletion
D010446:Peptide Fragments; D006639:Histidine; D006641:Histidinol; D000217:Acyltransferases; C050480:glycylpeptide N-tetradecanoyltransferase; D017868:Cyclic AMP-Dependent Protein Kinases; D004047:Diethyl Pyrocarbonate