Requirement for GLY-60 of Escherichia coli adenylyl cyclase for ATP binding and catalytic activity.
The region of Escherichia coli adenylyl cyclase spanned by glycine-55 to threonine-65 was tested for its importance for enzyme activity. Site-directed mutagenesis was used to replace glycine-55 and glycine-60 as well as lysine-59, leucine-63 and threonine-65 with other amino acids. While substitution of glycine-55 with aspartic acid produced no significant change in kinetic parameters, the change of glycine-60 to aspartic acid or asparagine eliminated binding to 8-azido-ATP and decreased the Vmax (two orders of magnitude) and Km (factor of four-five). Smaller effects on kinetic parameters were observed with substitutions of lysine-59, leucine-63 or threonine-65.
Amin
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Laboratory of Biochemical Genetics, National Heart, Lung and Blood Institute, Bethesda, MD 20892.
Peterkofsky
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A
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D000262:Adenylyl Cyclases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D001386:Azides / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005998:Glycine; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping
D001386:Azides; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; C017040:8-azidoadenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D000262:Adenylyl Cyclases; D005998:Glycine