PATTY ACYL TRANSFER BY HUMAN N-MYRISTYL TRANSFERASE IS DEPENDENT UPONCONSERVED CYSTEINE AND HISTIDINE-RESIDUES

N-Myristyl transferase (Nmt) catalyzes attachment of myristate onto th e N terminus of suitable proteins. In order to identify amino acids im portant for catalytic functions, human Nmt and mutants representing al l six conserved cysteine and histidine residues (Cys-169, Cys-214, His -131, His-171, His-218, and His-293) were expressed in Escherichia coi l 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 substr ate protein, pp60(v-src), the mutants showed differential incorporatio n of radiolabeled myristic acid into v-Src protein. In vitro experimen ts monitoring transfer of myristyl CoA to a peptide homologous to the N terminus of pp60(v-src) gave results similar to those obtained in vi vo. Our studies showed that mutation at Cys-169, His-171, and especial ly His-293 interfered with formation of an acyl-enzyme intermediate, w hile human Nmts containing mutations at Cys-169, His-218, or His-293 s howed greatly attenuated abilities to form acylated product. We propos e a model for the Nmt reaction mechanism in which Cys-169 sen es as th e fatty acid attachment site for a covalent myristyl enzyme intermedia te, while His-171 acts as a general acid/base and His-293 as a specifi c acid/base during acyl-enzyme intermediate formation. His-218 could t hen act as an acid or base needed to catalyze transfer of the acyl gro up from the acyl enzyme intermediate to a polypeptide substrate. This working model will be useful for the design of regulators of Nmt funct ion.