Purification and characterization of aminoglycoside 3'-phosphotransferase type IIa and kinetic comparison with a new mutant enzyme.
Aminoglycoside 3'-phosphotransferase [APH(3')s] provide an important means for high-level resistance to neomycin- and kanamycin-type aminoglycoside antibiotics. A four-step purification which affords milligram quantities of homogeneous APH(3') type IIa [APH(3')-IIa] is described. The kinetic parameters for the turnover of five substrates by the enzyme were determined, and the pH dependence and metal activation for catalysis were investigated. All five cysteines in the amino acid sequence of the enzyme exist in their reduced forms; hence, there are no disulfide bonds in the protein. Modification of the cysteine thiols by S-cyanylation showed essentially no effect on the enzymatic activity. A mutant enzyme derived from APH-3'-IIa, which possesses a conservative Glu-182-Asp point mutation and which provides diminished resistance to G418 (R. L. Yenofsky, M. Fine, and J. W. Pellow, Proc. Natl. Acad. Sci. USA 87:3435-3439, 1990), was also purified to homogeneity. Kinetic analysis of this mutant protein indicated an increase of approximately ninefold in the Km for Mg2+ ATP. Insofar as Km may approximate Ks, this finding argues for the involvement of residue 182 in the binding of Mg2+ ATP. Thus, purified APH(3')-IIa and a point mutant derivative enzyme were characterized enzymologically, and the roles of metal cofactors and the five reduced cysteine residues were probed in the wild-type enzyme.
Department of Chemistry, Wayne State University, Detroit, Michigan 48202.
J J
JJ
Siregar
S A
SA
Lerner
S
S
Mobashery
D000596:Amino Acids / Q000032:analysis; D002852:Chromatography, Ion Exchange; D003545:Cysteine / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D019868:Kanamycin Kinase; D007700:Kinetics; D008670:Metals / Q000378:metabolism; D008970:Molecular Weight; D009154:Mutation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D013050:Spectrometry, Fluorescence
D000596:Amino Acids; D008670:Metals; D017853:Phosphotransferases (Alcohol Group Acceptor); D019868:Kanamycin Kinase; D003545:Cysteine