The negative charge of Glu-127 in protein kinase A and its biorecognition.
A set of mutants of protein kinase A (PKA) in which Gln-127 was replaced by Gln, Asp, Asn, and Arg was prepared. Their Km and Vmax values show that the negative charge of Glu-127 (not merely its hydrogen bonding capacity) is indispensable for the kinase activity, since Glu-127/Gln is inactive, in spite of the fact that it can form hydrogen bonds and is very similar in bulkiness and conformation to wt-PKA. Glu-127 is involved in the biorecognition of PKA, interacting ionically with the positively charged guanido group of Arg P-3 (a major recognition element in the consensus sequence of PKA). In support of this conclusion, it is shown that a regression of the Glu-127 carboxylate by 1.54 A (as in Glu-127/Asp) results in an active kinase with a similar thermal stability and susceptibility to conformation-dependent proteolysis, a similar Vmax, an identical Km for ATP, but a > 20-fold higher Km for kemptide. The two inactive mutants of PKA, Glu-127/Gln and Glu-127/Asn, are potentially useful for studying protein-protein interactions of PKA, e.g. for monitoring enzymatically the displacement of active PKA from its complexes.
Batkin
M
M
Department of Biological Regulation, The Weizmann Institute of Science, Rehovot, Israel.
Shaltiel
S
S
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D016384:Consensus Sequence; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry* / Q000378:metabolism*; D004795:Enzyme Stability; D018698:Glutamic Acid; D006358:Hot Temperature; D006860:Hydrogen Bonding; D007700:Kinetics; D046911:Macromolecular Substances; D051379:Mice; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013816:Thermodynamics; D008625:Tiopronin / Q000378:metabolism
D046911:Macromolecular Substances; D011994:Recombinant Proteins; D018698:Glutamic Acid; D000255:Adenosine Triphosphate; D008625:Tiopronin; D017868:Cyclic AMP-Dependent Protein Kinases