Li H, Jordan F
Department of Chemistry, Program in Cellular and Molecular Biodynamics, Rutgers, the State University, Newark, New Jersey 07102, USA.
Biochemistry. 1999 Aug 3;38(31):10004-12. doi: 10.1021/bi9902440.
Oligonucleotide-directed site-specific mutagenesis was carried out on pyruvate decarboxylase (EC 4.1.1.1) from Saccharomyces cerevisiae at W412, located on the putative substrate activation pathway and linking E91 on the alpha domain with W412 on the gamma domain of the enzyme. While C221 on the beta domain is the residue at which substrate activation is triggered [Baburina, I., et al. (1994) Biochemistry 33, 5630-5635; Baburina, I., et al. (1996) Biochemistry 35, 10249-10255], that information, via the substrate bound at C221, is transmitted to H92 on the alpha domain, across the domain divide from C221 [Baburina, I., et al. (1998) Biochemistry 37, 1235-1244; Baburina, I., et al. (1998) Biochemistry 37, 1245-1255], thence to E91 on the alpha domain [Li, H., and Jordan, F. (1999) Biochemistry 38, 9992-10003], and then on to W412 on the gamma domain and to the active site thiamin diphosphate located at the interface of the alpha and gamma domains [Arjunan, D., et al. (1996) J. Mol. Biol. 256, 590-600]. Substitution at W412 with F and A was carried out, resulting in active enzymes with specific activities about 4- and 10-fold lower than that of the wild-type enzyme. Even though W412 interacts with E91 and H115 via a main chain hydrogen bond donor and acceptor, respectively, there is clear evidence for the importance of the indole side chain of W412 from a variety of experiments: thermostability, fluorescence quenching, and the binding constants of the thiamin diphosphate, and circular dichroism spectroscopy, in addition to conventional steady-state kinetic measurements. While the substrate activation is still prominent in the W412F variant, its level is very much reduced in the W412A variant, signaling that the size of the side chain is also important in positioning the amino acids surrounding the active center to achieve substrate activation. The fluorescence studies demonstrate that W412 is a relatively minor contributor to the well-documented fluorescence of apopyruvate decarboxylase in its native state. The information about the W412 variants provides strong additional support for the putative substrate activation pathway from C221 --> H92 --> E91 --> W412 --> G413 --> thiamin diphosphate. The accumulating evidence for the central role of the beta domain in stabilizing the overall structure is summarized.
对来自酿酒酵母的丙酮酸脱羧酶（EC 4.1.1.1）位于假定底物激活途径上的W412进行了寡核苷酸定向位点特异性诱变，该位点将酶α结构域上的E91与γ结构域上的W412相连。虽然β结构域上的C221是触发底物激活的残基[巴布林娜，I.等人（1994年）《生物化学》33卷，5630 - 5635页；巴布林娜，I.等人（1996年）《生物化学》35卷，10249 - 10255页]，但该信息通过结合在C221上的底物，从C221跨越结构域分界线传递到α结构域上的H92[巴布林娜，I.等人（1998年）《生物化学》37卷，1235 - 1244页；巴布林娜，I.等人（1998年）《生物化学》37卷，1245 - 1255页]，再传递到α结构域上的E91[李，H.和乔丹，F.（1999年）《生物化学》38卷，9992 - 10003页]，然后传递到γ结构域上的W412以及位于α和γ结构域界面处的活性位点硫胺二磷酸[阿尔朱南，D.等人（1996年）《分子生物学杂志》256卷，590 - 600页]。用F和A替换W412，得到了活性酶，其比活性分别比野生型酶低约4倍和10倍。尽管W412分别通过主链氢键供体和受体与E91和H115相互作用，但通过各种实验有明确证据表明W412的吲哚侧链很重要：热稳定性、荧光猝灭、硫胺二磷酸的结合常数以及圆二色光谱，此外还有传统的稳态动力学测量。虽然在W412F变体中底物激活仍然显著，但其水平在W412A变体中大幅降低，这表明侧链大小在定位活性中心周围的氨基酸以实现底物激活方面也很重要。荧光研究表明，W412对天然状态下已充分记录的脱辅基丙酮酸脱羧酶荧光的贡献相对较小。关于W412变体的信息为从C221→H92→E91→W412→G4l3→硫胺二磷酸的假定底物激活途径提供了有力的额外支持。总结了β结构域在稳定整体结构中核心作用的越来越多的证据。
