Cellini Barbara, Bertoldi Mariarita, Montioli Riccardo, Borri Voltattorni Carla
Dipartimento di Scienze Neurologiche e della Visione, Sezione di Chimica Biologica, Facoltà di Medicina e Chirurgia, Università degli Studi di Verona, Strada Le Grazie, 8, 37134 Verona, Italy.
Biochemistry. 2005 Oct 25;44(42):13970-80. doi: 10.1021/bi051433n.
Tyr 64, hydrogen-bonded to coenzyme phosphate in Treponema denticola cystalysin, was changed to alanine by site-directed mutagenesis. Spectroscopic and kinetic properties of the Tyr 64 mutant were investigated in an effort to explore the differences in coenzyme structure and kinetic mechanism relative to those of the wild-type enzyme. The wild type displays coenzyme absorbance bands at 418 and 320 nm, previously attributed to ketoenamine and substituted aldamine, respectively. The Tyr 64 mutant exhibits absorption maxima at 412 and 325 nm. However, the fluorescence characteristics of the latter band are consistent with its assignment to the enolimine form of the Schiff base. pK(spec) values of approximately 8.3 and approximately 6.5 were observed in a pH titration of the wild-type and mutant coenzyme absorbances, respectively. Thus, Tyr 64 is probably the residue involved in the nucleophilic attack on C4' of pyridoxal 5'-phosphate (PLP) in the internal aldimine. Although the Tyr 64 mutant exhibits a lower affinity for PLP and lower turnover numbers for alpha,beta-elimination and racemization than the wild type, the pH profiles for their Kd(PLP) and kinetic parameters are very similar. Rapid scanning stopped-flow and chemical quench experiments suggest that, in contrast to the wild type, for which the rate-determining step of alpha,beta-elimination of beta-chloro-L-alanine is the release of pyruvate, the rate-determining step for the mutant in the same reaction is the formation of alpha-aminoacrylate. Altogether, these results provide new insights into the catalytic mechanism of cystalysin and highlight the functional role of Tyr 64.
在齿垢密螺旋体溶胞素中与辅酶磷酸形成氢键的酪氨酸64通过定点诱变被替换为丙氨酸。对酪氨酸64突变体的光谱和动力学性质进行了研究，以探索其辅酶结构和动力学机制与野生型酶的差异。野生型在418和320nm处显示辅酶吸收带，之前分别归因于酮烯胺和取代醛胺。酪氨酸64突变体在412和325nm处呈现吸收最大值。然而，后一个吸收带的荧光特性与其被指定为席夫碱的烯醇亚胺形式一致。在对野生型和突变体辅酶吸收进行pH滴定过程中，分别观察到约8.3和约6.5的pK(spec)值。因此，酪氨酸64可能是参与对内部醛亚胺中磷酸吡哆醛（PLP）的C4'进行亲核攻击的残基。尽管酪氨酸64突变体对PLP的亲和力较低，且α,β-消除和消旋作用的周转数低于野生型，但其Kd(PLP)和动力学参数的pH曲线非常相似。快速扫描停流和化学淬灭实验表明，与野生型不同，野生型β-氯-L-丙氨酸α,β-消除的速率决定步骤是丙酮酸的释放，而突变体在同一反应中的速率决定步骤是α-氨基丙烯酸酯的形成。总之，这些结果为溶胞素的催化机制提供了新的见解，并突出了酪氨酸64的功能作用。
