De Bolle X, Vinals C, Prozzi D, Paquet J Y, Leplae R, Depiereux E, Vandenhaute J, Feytmans E
Département de Biologie, Facultés Universitaires Notre-Dame de la Paix, Namur, Belgium.
Eur J Biochem. 1995 Jul 1;231(1):214-9. doi: 10.1111/j.1432-1033.1995.tb20689.x.
The lack of crystal structure for tetrameric yeast alcohol dehydrogenases (ADHs) has precluded, until now, the identification of the residues involved in subunit contacts. In order to address this question, we have characterized the thermal stability and dissociation propensity of native ADH I and ADH II isozymes as well as of several chimeric (ADH I-ADH II) enzymes. Three groups of substitutions affecting the thermostability have been identified among the 24 substitutions observed between isozymes I and II. The first group contains a Cys277-->Ser substitution, located at the interface between subunits in a three-dimensional model of ADH I, based on the crystallographic structure of the dimeric horse liver ADH. In the second group, the Asp236-->Asn substitution is located in the same interaction zone on the model. The stabilizing effect of this substitution can result from the removal of a charge repulsion between subunits. It is shown that the effect of these two groups of substitutions correlates with changes in dissociation propensities. The third group contains the Met168-->Arg substitution that increases the thermal stability, probably by the formation of an additional salt bridge between subunits through the putative interface. These data suggest that at least part of the subunit contacts observed in horse liver ADH are located at homologous positions in yeast ADHs.
直到现在，由于缺乏四聚体酵母乙醇脱氢酶（ADHs）的晶体结构，阻碍了对参与亚基接触的残基的鉴定。为了解决这个问题，我们对天然ADH I和ADH II同工酶以及几种嵌合（ADH I-ADH II）酶的热稳定性和解离倾向进行了表征。在同工酶I和II之间观察到的24个取代中，已鉴定出三组影响热稳定性的取代。第一组包含一个Cys277→Ser取代，基于二聚体马肝ADH的晶体结构，该取代位于ADH I三维模型中亚基之间的界面处。在第二组中，Asp236→Asn取代位于模型上的同一相互作用区域。该取代的稳定作用可能源于亚基之间电荷排斥的消除。结果表明，这两组取代的效果与解离倾向的变化相关。第三组包含Met168→Arg取代，该取代可能通过假定的界面在亚基之间形成额外的盐桥来提高热稳定性。这些数据表明，在马肝ADH中观察到的至少部分亚基接触位于酵母ADHs的同源位置。
