Purification and properties of the apple fruit ethylene-forming enzyme.
The enzyme that oxidatively converts 1-aminocyclopropanecarboxylic acid (ACC) to ethylene, a key plant growth hormone, has been classified, on the basis of a comparison of homologous protein sequences (derived from the cDNA sequences), as a member of a family of non-heme iron proteins that includes plant and bacterial oxidative enzymes. This knowledge has facilitated the purification of the relatively abundant ethylene-forming enzyme to homogeneity from apple tissue. The properties of the enzyme are consistent with two other recent reports that describes its purification by different protocols, lending credence to the assertion that the key protein has been isolated. New characterizations of the protein have been conducted. Electrospray mass spectrometry shows that its molecular weight (35 331.8 +/- 5 amu) is approximately 50 amu higher than that predicted from the cDNA sequence, identifying the blocking group at the N-terminus as acetyl. The enzyme is activated by bicarbonate at low concentration but is inhibited at high concentration, with the maximum activation occurring at 5 mM. The iron concentration leading to half-maximal activity is 1 microM. The enzyme self-inactivates during turnover. The availability of the purified enzyme will permit definitive studies of the mechanism by which ethylene is produced and provide opportunities to discover molecules that inhibit the process.
Pirrung
M C
MC
P. M. Gross Chemical Laboratory, Department of Chemistry, Duke University, Durham, North Carolina 27708-0346.
Kaiser
L M
LM
Chen
J
J
D000595:Amino Acid Sequence; D001639:Bicarbonates / Q000494:pharmacology; D002245:Carbon Dioxide / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D005030:Ethylenes / Q000378:metabolism*; D005638:Fruit / Q000201:enzymology*; D007700:Kinetics; D008190:Lyases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D013058:Mass Spectrometry; D008667:Metalloproteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D019159:Nonheme Iron Proteins; D017386:Sequence Homology, Amino Acid
D001639:Bicarbonates; D005030:Ethylenes; D008667:Metalloproteins; D019159:Nonheme Iron Proteins; D002245:Carbon Dioxide; C036216:ethylene; D008190:Lyases; C034693:ethylene forming enzyme