Distinguishing among protein kinases by substrate specificities.
In the previous paper, N-methylated peptides were shown to be sensitive probes of substrate conformation within the adenosine cyclic 3',5'-phosphate dependent protein kinase (A-kinase) active site. While it has been shown that other protein kinases will catalyze the phosphorylation of the same peptide sequences as A-kinase, there is as yet little information as to whether the protein kinases differentiate between substrates on the basis of conformation. For this reason, the conformationally restricted N-methylated peptides were used to probe the active site of guanosine cyclic 3',5'-phosphate dependent protein kinase (G-kinase), which is homologous in sequence to [Takio, K., Wade, R. D., Smith, S. B., Krebs, E. G., Walsh, K. A., & Titani, K. (1984) Biochemistry 23, 4207-4218] and which has substrate specificities similar to [Lincoln, T. M., & Corbin, J. D. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 3239-3243] those of A-kinase. Although this enzyme appears to bind the peptides in a conformation resembling that of conformation A, it is more able to accommodate backbone methylation than is A-kinase. A peptide substrate at least 700-fold selective for G-kinase over A-kinase was found. Backbone methylation may, therefore, represent a way of making peptide substrates and inhibitors selective for a particular kinase.
Thomas
N E
NE
Laboratory of Bioorganic Chemistry and Biochemistry, Rockefeller University, New York, New York 10021.
Bramson
H N
HN
Nairn
A C
AC
Greengard
P
P
Fry
D C
DC
Mildvan
A S
AS
Kaiser
E T
ET
D000818:Animals; D001665:Binding Sites; D007700:Kinetics; D008745:Methylation; D011485:Protein Binding; D011487:Protein Conformation; D011494:Protein Kinases / Q000378:metabolism*; D013379:Substrate Specificity
D011494:Protein Kinases