Role of the glycine triad in the ATP-binding site of cAMP-dependent protein kinase.
A glycine-rich loop in the ATP-binding site is one of the most highly conserved sequence motifs in protein kinases. Each conserved glycine (Gly-50, Gly-52, and Gly-55) in the catalytic (C) subunit of cAMP-dependent protein kinase (cAPK) was replaced with Ser and/or Ala. Active mutant proteins were expressed in Escherichia coli, purified to apparent homogeneity, separated into phosphoisoforms, and characterized. Replacing Gly-55 had minimal effects on steady-state kinetic parameters, whereas replacement of either Gly-50 or Gly-52 had major effects on both Km and kcat values consistent with the prediction of the importance of the tip of the glycine-rich loop for catalysis. Substitution of Gly-50 caused a 5-8-fold reduction in Km (ATP), an 8-12-fold increase in Km (peptide), and a 3-5-fold drop in kcat. The Km (ATP) and Km (peptide) values of C(G52S) were increased 8- and 18-fold, respectively, and the kcat was decreased 6-fold. In contrast to catalytic efficiency, the ATPase rates of C(G50S) and C(G52S) were increased by more than an order of magnitude. The thermostability of each mutant was slightly increased. Unphosphorylated C(G52S) was characterized as well as several isoforms phosphorylated at a single site, Ser-338. All of these phosphorylation-defective mutants displayed a substantial decrease in both enzymatic activity and thermal stability that correlated with the missing phosphate at Thr-197. These results are correlated with the crystal structure, models of the respective mutant proteins, and conservation of the Glys within the protein kinase family.
Hemmer
W
W
Swiss Federal Office of Public Health, Division of Food Science, Section of Microbiology and Hygiene, CH-3003 Bern, Switzerland.
McGlone
M
M
Tsigelny
I
I
Taylor
S S
SS
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000409:Alanine / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D005998:Glycine / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D012694:Serine / Q000378:metabolism; D013237:Stereoisomerism; D013696:Temperature
D012694:Serine; D000255:Adenosine Triphosphate; D017868:Cyclic AMP-Dependent Protein Kinases; D000251:Adenosine Triphosphatases; D000409:Alanine; D005998:Glycine