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首页> 外文期刊>Biochemistry >Kinetic and Mutagenic Characterization of the Chromosomally Encolded Salmonella enterica AAC(6')-Iy Aminoglycoside N-Acetyltransferase
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Kinetic and Mutagenic Characterization of the Chromosomally Encolded Salmonella enterica AAC(6')-Iy Aminoglycoside N-Acetyltransferase

机译：肠溶沙门氏菌AAC（6'）-Iy氨基糖苷N-乙酰基转移酶的染色体的动力学和诱变特性。

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The chromosomally encoded aminoglycoside N-acetyltransferase, AAC(6')-Iy, from Salmonella enterica confers resistance toward a number of aminoglycoside antibiotics. The structural gene was cloned and expressed and the purified enzyme existed in solution as a dimer of ca. 17 000 Da monomers. Acetyl- CoA was the preferred acyl donor, and most therapeutically important aminoglycosides were substrates for acetylation. Exceptions are those aminoglycosides that possess a 6'-hydroxyl substituent (e.g., lividomycin). Thus, the enzyme exhibited regioselective and exclusive acetyltransferase activity to 6'- amine-containing aminoglycosides. The enzyme exhibited Michaelis-Menten kinetics for some amino- glycoside substrates but "substrate activation" with others. Kinetic studies supported a random kinetic mechanism for the enzyme. The enzyme was inactivated by iodoacetamide in a biphasic manner, with half of the activity being lost rapidly and the other half more slowly. Tobramycin, but not acetyl-CoA, protected against inactivation. Each of the three cysteine residues (C70, C109, C145) in the wild-type enzyme were carboxamidomethylated by iodoacetamide. Cysteine 109 in AAC(6')-Iy is conserved in 12 AAC( 6') enzyme sequences of the major class I subfamily. Surprisingly, mutation of this residue to alanine neither abolished activity nor altered the biphasic inactivation by iodoacetamide. The maximum velocity and V/K values for a number of aminoglycosides were elevated in this single mutant, and the kinetic behavior of substrates exhibiting linear vs nonlinear kinetics was reversed. Cysteine 70 in AAC(6')-Iy is either a cysteine or a threonine residue in all 12 AAC(6') enzymes of the major class I subfamily. The double mutant, C109A/C70A, was not inactivated by iodoacetamide. The double mutant exhibited large increases in the Km values for both acetyl-CoA and aminoglycoside substrates, and all aminoglycoside substrates exhibited Michaelis-Menten kinetics. Solvent kinetic isotope~ffects on V/K were normal for the WT enzyme and inverse for the double mutant. We discuss a chemical mechanism and the likely rate-limiting steps for both the wild-type and mutant forms of the enzyme.
机译：来自肠沙门氏菌的染色体编码的氨基糖苷N-乙酰基转移酶AAC（6'）-Iy赋予了对多种氨基糖苷抗生素的抗性。克隆并表达了结构基因，纯化的酶以约二聚体形式存在于溶液中。 17000 Da单体。乙酰辅酶A是优选的酰基供体，最重要的治疗性氨基糖苷是乙酰化的底物。具有6'-羟基取代基的那些氨基糖苷（例如lividomycin）是例外。因此，该酶对含6'-胺的氨基糖苷表现出区域选择性和排他的乙酰转移酶活性。该酶对某些氨基糖苷底物表现出Michaelis-Menten动力学，但对其他一些则表现出“底物激活”。动力学研究支持该酶的随机动力学机制。碘乙酰胺以双相方式使该酶失活，其中一半的活性迅速丧失，而另一半则更缓慢。妥布霉素而非乙酰辅酶A可以防止失活。野生型酶中的三个半胱氨酸残基（C70，C109，C145）均被碘乙酰胺羧酰胺甲基化。 AAC（6'）-Iy中的半胱氨酸109在主要I类亚家族的12个AAC（6'）酶序列中保守。令人惊讶地，该残基突变为丙氨酸既不消除活性也不改变碘乙酰胺的双相失活。在此单个突变体中，许多氨基糖苷的最大速度和V / K值都提高了，并且显示线性动力学与非线性动力学的底物的动力学行为相反。 AAC（6'）-Iy中的半胱氨酸70是主要I类亚家族的所有12种AAC（6'）酶中的半胱氨酸或苏氨酸残基。双重突变体C109A / C70A没有被碘乙酰胺灭活。对于乙酰辅酶A和氨基糖苷底物，双突变体均显示出Km值的大幅增加，并且所有氨基糖苷底物均表现出Michaelis-Menten动力学。对于WT酶，溶剂动力学同位素对V / K的影响是正常的，而对于双突变体则是相反的。我们讨论了酶的野生型和突变体形式的化学机制和可能的限速步骤。

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