Lys-Ala mutations of type I adenylyl cyclase result in altered susceptibility to inhibition by adenine nucleoside 3'-polyphosphates.
Native and recombinant wild type and mutant forms of type I adenylyl cyclase, expressed in fall army worm ovarian cells (Sf9) cells, with mutations Lys-923-Ala, Lys-921-Ala, and Lys-350-Ala, retained the characteristic noncompetitive inhibition by adenine nucleoside 3'-polyphosphates, but exhibited substantially different sensitivities to inhibition by them. The type I K923A enzyme resulted in increased IC(50) values, e.g., >100-fold for 2'-deoxyadenosine-3'-monophosphate, but the shift diminished as the number of 3'-phosphates increased. The K921A mutation increased IC(50) values approximately 5-fold for all adenine nucleosides tested, whereas the K350A mutation increased IC(50) values approximately 6- to 8-fold for all adenine nucleosides tested except 2'-deoxyadenosine-3'-diphosphate, which was increased >/=2-fold. The data suggest that 3'-phosphates sufficiently increase binding affinity of these ligands to compensate for the reduced coordination of the adenine moiety induced by the K923A mutation. Moreover, the altered structures induced by both K350A and K921A mutations impair ligand binding in general, but paradoxically those resulting from the K350A change minimally affected nucleoside 3'-diphosphate binding, implying that selective changes in ligand binding can be induced by this site-specific mutation.
Shoshani
I
I
Department of Physiology and Biophysics, State University of New York, Stony Brook, New York 11794-8661, USA.
Bianchi
G
G
Désaubry
L
L
Dessauer
C W
CW
Johnson
R A
RA
D000227:Adenine Nucleotides / Q000494:pharmacology*; D000067956:Adenylyl Cyclase Inhibitors; D000262:Adenylyl Cyclases / Q000737:chemistry / Q000378:metabolism*; D000409:Alanine; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001921:Brain / Q000201:enzymology; D002460:Cell Line; D017124:Conserved Sequence; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005260:Female; D007700:Kinetics; D008239:Lysine; D008969:Molecular Sequence Data; D010053:Ovary; D010446:Peptide Fragments / Q000737:chemistry; D051381:Rats; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D018411:Spodoptera; D014162:Transfection
D000227:Adenine Nucleotides; D000067956:Adenylyl Cyclase Inhibitors; D004791:Enzyme Inhibitors; D010446:Peptide Fragments; D011994:Recombinant Proteins; D000262:Adenylyl Cyclases; D008239:Lysine; D000409:Alanine