Altered substrate specificity by substitutions at Tyr218 in bacterial aminoglycoside 3'-phosphotransferase-II.
Mutant aminoglycoside 3'-phosphotransferase II enzymes were produced in which Tyr218 was changed to serine, aspartic acid, or phenylalanine. In each case the mutation resulted in increased bacterial susceptibility to neomycin and kanamycin, while simultaneously increasing the Km values for these substrates. For the Ser and Asp mutants, bacterial resistance to amikacin increased, with a concomitant increase in affinity for this drug. Initial velocity studies indicated that the wild-type and mutant enzymes all followed Michaelis-Menten kinetics. Although these mutagenic substitutions changed the substrate specificity of these enzymes they did not alter the enzyme affinity for Mg(2+)-ATP.
Kocabiyik
S
S
Department of Biology, University of Louisville, KY 40292.
Perlin
M H
MH
D000595:Amino Acid Sequence; D001483:Base Sequence; D004352:Drug Resistance, Microbial / Q000235:genetics; D019868:Kanamycin Kinase; D007613:Kanamycin Resistance / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009355:Neomycin / Q000494:pharmacology*; D010770:Phosphotransferases / Q000235:genetics* / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D010770:Phosphotransferases; D019868:Kanamycin Kinase; D009355:Neomycin