Ethylene is a gaseous hormone that regulates a wide range of physiological responses in plants. The terminal enzyme in the biosynthetic pathway, 1-aminocyclopropane-1-carboxylate (ACC) oxidase, has recently been shown to be activated by CO2. Only one other enzyme has been shown to be activated by CO2 (ribulosebisphosphate carboxylase) via carbamate formation on the ?-amino group of a lysyl residue. By analogy, we are investigating whether a specific lysyl residue is modified by CO2 during activation. Because the carbamate is extremely labile, it will be necessary to form a stable methyl ester with the carbamate to allow proteolytic digestion while maintaining the carbamate. After proteolysis, the peptides will be analyzed by MALDI-MS to detect differences between the nonactivated and the CO2-activated enzyme. The enzyme has been purified after overexpression in E. coli. The purified form is estimated to be only 2-5% activated, and it can be fully activated by CO2. The mass spectrometry analysis will be crucial to determining which of the 26 absolutely conserved lysine residues is modified by CO2. We will then pursue site-directed mutagenesis and x-ray crystallography to confirm our results.

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Biotechnology Resource Grants (P41)
Project #
3P41RR000480-28S1
Application #
6258826
Study Section
Project Start
1997-06-01
Project End
1999-11-30
Budget Start
1998-10-01
Budget End
1999-09-30
Support Year
28
Fiscal Year
1999
Total Cost
Indirect Cost
Name
Michigan State University
Department
Type
DUNS #
193247145
City
East Lansing
State
MI
Country
United States
Zip Code
48824
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