Characterization of a polyhistidine-tagged form of human myristoyl-CoA: protein N-myristoyltransferase produced in Escherichia coli.
The enzyme myristoyl-CoA:protein N-myristoyltransferase is responsible for the attachment of a myristoyl group to the N-terminal glycine of a number of cell, viral and fungal proteins. In order to overcome the difficulties of purification of this enzyme from tissue sources, we have produced an N-terminally polyhistidine-tagged version of the enzyme and expressed this in Escherichia coli. The resulting enzyme has a molecular mass of 53 kDa and is fully active showing the expected specificity for myristic acid and causing the N-terminal myristoylation of both synthetic peptide and protein substrates in vitro. The enzyme exhibits a broad pH optimum peaking at a pH of 8.0 and has a Km for myristoyl-CoA of 7.6 microM. The two synthetic peptide substrates based on the N-terminal sequence of the catalytic subunit of protein kinase A (GNAAAARR) and of p60src (GSSKSKPKDPSQRRRY) have different kinetic parameters with Km values of 115.2 microM and 44.2 microM and Vmax values of 95 and 120 nmol.min-1.mg-1, respectively. The expressed enzyme is partially inhibited (50%) by iodoacetamide at 5 mM and fully inhibited by diethylpyrocarbonate at 10 mM. This latter inhibition can be prevented by including histidine in the incubation of the enzyme and inhibitor. Antisera raised to synthetic peptides based on sequences derived from the N- and C- terminus of the human enzyme reacted with the expressed protein on Western blots, but only the N-terminal sequence reacted with the native protein suggesting that the C-terminus may be not be accessible. The enzyme can catalyse the removal of a myristoyl group from myristoylated peptides but does so only in the presence of added coenzyme A.
McIlhinney
R A
RA
Medical Research Council Anatomical Neuropharmacology Unit, Oxford, England.
Patel
P B
PB
McGlone
K
K
D000215:Acylation; D000217:Acyltransferases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D017931:DNA Primers; D004047:Diethyl Pyrocarbonate; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D005227:Fatty Acids / Q000737:chemistry; D006639:Histidine; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007460:Iodoacetamide; D007525:Isoelectric Focusing; D007700:Kinetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry*; D011994:Recombinant Proteins; D013379:Substrate Specificity
D017931:DNA Primers; D005227:Fatty Acids; D010455:Peptides; D011994:Recombinant Proteins; C033223:polyhistidine; D006639:Histidine; D000217:Acyltransferases; C050480:glycylpeptide N-tetradecanoyltransferase; D004047:Diethyl Pyrocarbonate; D007460:Iodoacetamide