Mauch L, Bichler V, Brandsch R
Biochemisches Institut, Universität Freiburg, FRG.
FEBS Lett. 1989 Oct 23;257(1):86-8. doi: 10.1016/0014-5793(89)81792-2.
In 6-hydroxy-D-nicotine oxidase (6-HDNO) FAD is covalently bound to His71 of the polypeptide chain by an 8 alpha-(N3-histidyl)-riboflavin linkage. The FAD-binding histidine was exchanged by site-directed mutagenesis to either a Cys- or Tyr-residue, two amino acids known to be involved in covalent binding of FAD in other enzymes, or to a Ser-residue. None of the amino acid replacements for His71 allowed covalent FAD incorporation into the 6-HDNO polypeptide. Thus, the amino acid residues involved in covalent FAD-binding require a specific polypeptide surrounding in order for this modification to proceed and cannot be replaced with each other. Enzyme activity was completely abolished with Tyr in place of His71. 6-HDNO activity with non-covalently bound FAD was found with 6-HDNO-Cys and to a lesser extent also with 6-HDNO-Ser. However, the Km values for 6-HDNO-Cys and 6-HDNO-Ser were increased approximately 20-fold as compared to 6-HDNO-His. Both mutant enzymes, in contrast to the wild-type enzyme, needed additional FAD in the enzymatic assay (50 microM for 6-HDNO-Ser and 10 microM for 6-HDNO-Cys) for maximal enzyme activity.
在6-羟基-D-尼古丁氧化酶（6-HDNO）中，黄素腺嘌呤二核苷酸（FAD）通过8α-（N3-组氨酰基）-核黄素键与多肽链的His71共价结合。通过定点诱变将FAD结合组氨酸替换为半胱氨酸残基或酪氨酸残基，这两种氨基酸已知参与其他酶中FAD的共价结合，或者替换为丝氨酸残基。His71的氨基酸替换均未使FAD共价掺入6-HDNO多肽中。因此，参与FAD共价结合的氨基酸残基需要特定的多肽环境才能进行这种修饰，并且不能相互替代。用酪氨酸取代His71时，酶活性完全丧失。在6-HDNO-Cys中发现了与非共价结合FAD的6-HDNO活性，在6-HDNO-Ser中也发现了程度较低（的该活性）。然而，与6-HDNO-His相比，6-HDNO-Cys和6-HDNO-Ser的米氏常数（Km值）增加了约20倍。与野生型酶相比，这两种突变酶在酶促测定中都需要额外的FAD（6-HDNO-Ser为50微摩尔，6-HDNO-Cys为10微摩尔）才能达到最大酶活性。
