Hevel J M, Mills S A, Klinman J P
Department of Chemistry and Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA.
Biochemistry. 1999 Mar 23;38(12):3683-93. doi: 10.1021/bi982199m.
The copper amine oxidases (CAOs) catalyze both the single-turnover modification of a peptidyl tyrosine to form the active-site cofactor 2,4,5-trihydroxyphenylalanine quinone (TPQ) and the oxidative deamination of primary amines using TPQ. The function of a strictly conserved tyrosine located within hydrogen-bonding distance to TPQ has been explored by employing site-directed mutagenesis on the enzyme from H. polymorpha to form the mutants Y305A, Y305C, and Y305F. Both Y305A and Y305C behave similarly with regard to aliphatic amine oxidase activity, showing 3-7-fold decreases in kinetic parameters relative to WT, while the more conservative substitution of Y305F results in a >100-fold decrease in kcat and >500-fold decrease in kcat/Km relative to WT for the reductive half-reaction. The oxidation of benzylamine by all three mutants is severely impaired, with very significant effects seen in the oxidative half-reaction. CAO activity was studied as a function of pH for WT and Y305A proteins. Profiles for WT-catalyzed methylamine oxidation and Y305A-catalyzed ethylamine oxidation are comparable, while profiles of Y305A-catalyzed methylamine oxidation suggest the pH-dependent build-up of an inhibitory intermediate, which was subsequently observed spectrophotometrically and is attributed to the product Schiff base. The relative effects of mutations at Y305 on catalytic turnover are, thus, concluded to be dependent on the nature of the amino acid which substitutes for tyrosine and the substrate used in amine oxidase assays. TPQ biogenesis experiments demonstrate a approximately 800-fold decrease in kobs for apo-Y305A compared to WT. Despite the strict conservation of Tyr305 in all CAOs, neither biogenesis nor catalytic turnover is abolished upon mutation of this residue. We propose an important, but nonessential, role for Tyr305 in the positioning of the TPQ precursor for biogenesis, and in the maintenance of the correct conformation for TPQ-derived intermediates during catalytic turnover.
铜胺氧化酶（CAOs）既能催化肽基酪氨酸的单轮修饰以形成活性位点辅因子2,4,5-三羟基苯丙氨酸醌（TPQ），又能利用TPQ催化伯胺的氧化脱氨反应。通过对多形汉逊酵母中的酶进行定点诱变，构建突变体Y305A、Y305C和Y305F，研究了一个与TPQ处于氢键距离内的严格保守酪氨酸的功能。Y305A和Y305C在脂肪族胺氧化酶活性方面表现相似，相对于野生型（WT），动力学参数降低了3至7倍，而Y305F这种更保守的取代导致还原半反应的kcat相对于WT降低了100倍以上，kcat/Km降低了500倍以上。所有这三个突变体对苄胺的氧化均受到严重损害，在氧化半反应中可见非常显著的影响。研究了野生型和Y305A蛋白的CAO活性与pH的关系。野生型催化甲胺氧化和Y305A催化乙胺氧化的曲线具有可比性，而Y305A催化甲胺氧化的曲线表明存在一种pH依赖性的抑制性中间体积累，随后通过分光光度法观察到该中间体，其归因于产物席夫碱。因此得出结论，Y305处突变对催化周转的相对影响取决于取代酪氨酸的氨基酸性质以及胺氧化酶测定中使用的底物。TPQ生物合成实验表明，与野生型相比，脱辅基Y3Y305A的kobs降低了约800倍。尽管在所有CAOs中Tyr305严格保守，但该残基突变后，生物合成和催化周转均未被消除。我们提出Tyr305在TPQ生物合成前体的定位以及催化周转过程中TPQ衍生中间体正确构象的维持方面起着重要但非必需的作用。
