Enzymatic properties of pierisin-1 and its N-terminal domain, a guanine-specific ADP-ribosyltransferase from the cabbage butterfly.
The cabbage butterfly, Pieris rapae, produces an ADP-ribosylating cytotoxic protein, pierisin-1. Unlike other ADP-ribosylating toxins, the acceptor site for ADP-ribosylation by pierisin-1 is the N-2 position of guanine bases in DNA. The present study was designed to characterize this novel guanine-specific ADP-ribosyltransferase, pierisin-1. The N-terminal polypeptide from Met-1 to Arg-233, but not the C-terminal Ser-234-Met-850 polypeptide, was found to exhibit guanine ADP-ribosyltransferase activity. Trypsin-treated pierisin-1, which is considered to be a "nicked" full-length form composed of associated N- and C-terminal fragments, also demonstrated such activity. Optimum conditions for the N-terminal polypeptide of pierisin-1 were pH 8-10, 37-40 degrees C, in the presence of 100-200 mM NaCl or KCl. Other metal ions such as Ca(2+) or Mg(2+) were not required. Kinetic studies demonstrated potent ADP-ribosyltransferase activity with a K(M) value for NAD of 0.17 mM and k(cat) of 55 per second. Under these optimum conditions, the specific activity of trypsin-treated pierisin-1 was about half (k(cat) = 25 per second). When the conditions were changed to pH 5-7 or 10-20 degrees C, some activity (6-55% or 5-20%, respectively, of that under optimal conditions) of the N-terminal polypeptide was still evident; however, almost all of the trypsin-treated enzyme activity disappeared. This implies the inhibition of the N-terminal enzyme domain by the associated C-terminal fragment. Long-term reactions indicated that a single molecule of pierisin-1 has the capacity to generate more than 10(6) ADP-ribosylated DNA adducts, which could cause the death of a mammalian cell.
Watanabe
Masahiko
M
Cancer Prevention Basic Research Project, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045. mwatanabe@gan2.ncc.go.jp
Enomoto
Shigeki
S
Takamura-Enya
Takeji
T
Nakano
Tsuyoshi
T
Koyama
Kotaro
K
Sugimura
Takashi
T
Wakabayashi
Keiji
K
D036002:ADP Ribose Transferases / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002080:Butterflies / Q000201:enzymology*; D004247:DNA / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D019476:Insect Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D009243:NAD / Q000378:metabolism; D009994:Osmolar Concentration; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D012313:RNA / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D014357:Trypsin / Q000378:metabolism
D018076:DNA, Complementary; D019476:Insect Proteins; D010446:Peptide Fragments; C113557:pierisin protein, insect; D009243:NAD; D012313:RNA; D004247:DNA; D036002:ADP Ribose Transferases; D014357:Trypsin