Site-directed random mutagenesis of AMP-binding residues in adenylate kinase.
Two highly conservative residues, Val67 and Gln101, in adenylate kinase are located in the hydrophobic region putatively involved in binding of the adenine ring of AMP. We have performed polymerase chain reaction-based random mutagenesis of the two residues using recombinant chicken muscle adenylate kinase cDNA as a template. The synthetic oligonucleotide primers contained A,G,C,T-mixed bases in the codons corresponding to those for Val67 and Gln101. The amplified fragments were ligated with the expression plasmid pKK223-3, and the mutant proteins expressed were identified by immunoblotting. Enzymatically active mutant proteins were selected on the basis of the growth at 45 degrees C of the temperature sensitive Escherichia coli mutant for adenylate kinase. At position 67, various amino acid residues other than Val have been found to restore the growth at 45 degrees C of the ts mutant. In contrast, only Gln, His, and Met could be present at position 101. These results are compatible with the proposal that Val67 contributes to the AMP binding through hydrophobic interactions and Gln101 by forming a hydrogen bond with the adenine ring. Indeed, several purified Val67 and Gln101 mutant enzymes exhibited markedly high Km values for AMP, whereas the Km values for MgATP were comparable to those of the wild-type enzyme. Substrate specificity for nucleoside monophosphates was changed significantly by the mutagenesis of the two residues.
Okajima
T
T
Institute of Scientific and Industrial Research, Osaka University.
Tanizawa
K
K
Fukui
T
T
D000249:Adenosine Monophosphate / Q000378:metabolism*; D000263:Adenylate Kinase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002645:Chickens; D018076:DNA, Complementary; D004926:Escherichia coli / Q000201:enzymology; D005973:Glutamine / Q000737:chemistry; D008969:Molecular Sequence Data; D009132:Muscles / Q000201:enzymology; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D011994:Recombinant Proteins; D013379:Substrate Specificity; D014633:Valine / Q000737:chemistry
D018076:DNA, Complementary; D011994:Recombinant Proteins; D005973:Glutamine; D000249:Adenosine Monophosphate; D000263:Adenylate Kinase; D014633:Valine