Fatty acyl transfer by human N-myristyl transferase is dependent upon conserved cysteine and histidine residues.
N-Myristyl transferase (Nmt) catalyzes attachment of myristate onto the N terminus of suitable proteins. In order to identify amino acids important for catalytic functions, human Nmt and mutants representing all six conserved cysteine and histidine residues (Cys-169, Cys-214, His-131, His-171, His-218, and His-293) were expressed in Escherichia coli and analyzed for their ability to bind and transfer myristic acid. N-Terminal histidine-tagged fusion proteins displayed varying abilities to form an association with radiolabeled myristic acid indicative of an acyl-enzyme intermediate. When co-expressed with an acceptor substrate protein, pp60v-src, the mutants showed differential incorporation of radiolabeled myristic acid into v-Src protein. In vitro experiments monitoring transfer of myristyl CoA to a peptide homologous to the N terminus of pp60 v-src gave results similar to those obtained in vivo. Our studies showed that mutation at Cys-169, His-171, and especially His-293 interfered with formation of an acyl-enzyme intermediate, while human Nmts containing mutations at Cys-169, His-218, or His-293 showed greatly attenuated abilities to form acylated product. We propose a model for the Nmt reaction mechanism in which Cys-169 serves as the fatty acid attachment site for a covalent myristyl enzyme intermediate, while His-171 acts as a general acid/base and His-293 as a specific acid/base during acyl-enzyme intermediate formation. His-218 could then act as an acid or base needed to catalyze transfer of the acyl group from the acyl-enzyme intermediate to a polypeptide substrate. This working model will be useful for the design of regulators of Nmt function.
Peseckis
S M
SM
Cell Biology and Genetics Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021.
Resh
M D
MD
D000217:Acyltransferases / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D017124:Conserved Sequence; D003545:Cysteine / Q000737:chemistry*; D004926:Escherichia coli / Q000201:enzymology; D005227:Fatty Acids / Q000378:metabolism*; D006639:Histidine / Q000737:chemistry*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides
D005227:Fatty Acids; D009838:Oligodeoxyribonucleotides; D006639:Histidine; D000217:Acyltransferases; C050480:glycylpeptide N-tetradecanoyltransferase; D003545:Cysteine