Poster: Enzymology

Abs # 243: Limited tryptic proteolysis of peptide deformylase generates a core protein that retains the majority of activity without a salt-requirement for solubility

Presenter:	Hanger, Katherine M., kmhang2@uky.edu
Authors	Hanger, Katherine M. (B) (A)  Houtz, Robert L. (D) (C)  Dirk, Lynnette M.A. (D) (C)
Affiliations:	(A): University of Kentucky (B): College of Biological Science (C): Plant Physiology/Biochemistry /Molecular Biology Program (D): Department of Horticulture

Peptide deformylase (DEF) catalyzes removal of the formyl group from the N termini of nascent polypeptides in both chloroplasts and mitochondria. The Arabidopsis peptide deformylase, AtDEF2, has relatively low sequence homology along with dissimilar kinetic parameters and pH dependence compared with E. coli DEF. Potentially interesting differences between DEF2 and the bacterial enzymes' active site environment could be revealed through structural studies of DEF2 but these investigations have been impeded by a concentration-dependent solubility requirement for sodium chloride. Limited tryptic proteolysis of DEF2 has been performed to generate a truncated form of the protein that requires less salt yet retains catalytic activity, as determined with aminopeptidase-linked activity assays. Proteolysis with 0.5 to 2% trypsin in 20 min left a catalytically active core with a mobility shift on a 8-16% gradient SDS-PAGE corresponding to a 3 kD loss in molecular mass. Direct N-terminal sequencing revealed one tryptic sensitive site at the N terminus accounting for a mass loss of 1.36 kD. Loss of a carboxy-terminal hexahistidyl sequence revealed by an absence of immunological reactivity to a hexahistidyl antibody identified the possibility of several carboxy-terminal tryptic sensitive sites. Identification of amino acid sequence for RP-HPLC purified carboxyl-terminal tryptic polypeptides as well as molecular mass determination by ES-MS will be used to identify the complete sequence of proteolyzed DEF.