Cysteine 42 is important for maintaining an integral active site for O-acetylserine sulfhydrylase resulting in the stabilization of the alpha-aminoacrylate intermediate.
Cysteine 42 is important for maintaining an integral active site for O-acetylserine sulfhydrylase resulting in the stabilization of the alpha-aminoacrylate intermediate.
复制标题
半胱氨酸 42 对于维持 O-乙酰丝氨酸硫化氢解酶的完整活性位点非常重要，从而导致 α-氨基丙烯酸酯中间体的稳定。
DOI：
10.1021/bi980647k
复制
发表时间：
1998
期刊：
影响因子：
--
通讯作者：
Cook,PF
中科院分区：
文献类型：
--
作者：
Tai,CH;Yoon,MY;Kim,SK;Rege,VD;Nalabolu,SR;Kredich,NM;Schnackerz,KD;Cook,PF
o -乙酰丝氨酸巯基水解酶-a （OASS-A）是一种依赖于吡哆醛5 ' -磷酸（PLP）的酶，来自于沙门氏菌，它能催化硫化物取代醋酸氨基乙酰丝氨酸（OAS）生成半胱氨酸。在席夫碱中α-氨基丙烯酸酯与活性位点PLP作为中间体的乒乓动力学机制中发生[Cook， P. F., Hara， S., Nalabolu， S. R., and Schnackerz， K. D.（1992）生物化学31,2298−2303]。Schiff碱基赖氨酸（K41）周围的序列已经确定[Rege， V. D, Kredich， N. M., Tai， C.-H.]， Karsten， W. E, Schnackerz， K. D, & Cook， P. F.（1996）生物化学35,13485−13493]，一级结构中唯一的半胱氨酸是赖氨酸的直接c端。为了评估C42的作用，通过定点诱变将其改变为丝氨酸和丙氨酸。根据紫外可见、荧光、远紫外和辅助因子诱导的CD和31p NMR研究，突变蛋白在结构上与野生型酶几乎相同，但注意到细微的结构差异。这两种突变蛋白的动力学特性与野生型酶有显著不同。与野生型相比，C42S突变体的OAS：乙酸裂解酶活性增加了50倍，半胱氨酸合成的inv降低了17倍，而C42A突变体的OAS：乙酸裂解酶活性降低了200倍，半胱氨酸合成的inv降低了30倍。然而，在这两种情况下，半胱氨酸合成和OAS：醋酸裂解酶活性产率的动力学参数的pH依赖性，在误差范围内，相同的pk值。在OASS-A的三维结构中，半胱氨酸42位于辅因子的后面，远离活性位点，指向蛋白质的内部。C42S和C42A突变蛋白中OASS-A的OAS：醋酸裂解酶活性的巨大变化可能是由于丝氨酸羟基（与半胱氨酸硫醇相比）向C42S中额外的亲水性氢键基团或远离C42A的亲水性基团的局部运动，重新定位了K41周围和包括K41在内的结构。K41的ε-氨基的微小移动可能改变PLP中氨基酸亲核位移的几何形状，导致α-氨基丙烯酸酯中间体的整体活性和稳定性发生变化。数据表明，单个氨基酸的取代只产生细微的结构变化，却能产生反应速率和整体机理的巨大差异。
O-Acetylserine sulfhydrylase-A (OASS-A) is a pyridoxal 5‘-phosphate (PLP) dependent enzyme fromSalmonellatyphimuriumthat catalyzes the β-replacement of acetate inO-acetyl-l-serine (OAS) by sulfide to givel-cysteine. The reaction occurs via a ping-pong kinetic mechanism in which α-aminoacrylate in Schiff base with the active site PLP is an intermediate [Cook, P. F., Hara, S., Nalabolu, S. R., and Schnackerz, K. D. (1992)Biochemistry 31, 2298−2303]. The sequence around the Schiff base lysine (K41) has been determined [Rege, V. D., Kredich, N. M., Tai, C.-H., Karsten, W. E., Schnackerz, K. D., & Cook, P. F. (1996)Biochemistry 35, 13485−13493], and the sole cysteine in the primary structure is immediately C-terminal to the lysine. In an effort to assess the role of C42, it has been changed to serine and alanine by site-directed mutagenesis. The mutant proteins are structurally nearly identical to the wild-type enzyme on the basis of UV−visible, fluorescence, far-UV and cofactor-induced CD, and31P NMR studies, but subtle structural differences are noted. Kinetic properties of both mutant proteins differ significantly from those of the wild-type enzyme. The C42S mutant exhibits a >50-fold increase in the OAS:acetate lyase activity and a 17-fold decrease inVfor the cysteine synthesis compared to the wild-type enzyme, while decreases of >200-fold in the OAS:acetate lyase activity and a 30-fold decrease inVfor the cysteine synthesis are found for the C42A mutant enzyme. In both cases, however, the pH dependence of kinetic parameters for cysteine synthesis and OAS:acetate lyase activity yield, within error, identical pKvalues. In the three-dimensional structure of OASS-A, cysteine 42 is located behind the cofactor, pointing away from the active site, toward the interior of the protein. The dramatic change in the OAS:acetate lyase activity of OASS-A in the C42S and C42A mutant proteins likely results from a localized movement of the serine hydroxyl (compared to the cysteine thiol) toward additional hydrophilic, hydrogen-bonding groups in C42S, or away from hydrophilic groups for C42A, repositioning structure around and including K41. Subtle movement of the ε-amino group of K41 may change the geometry for nucleophilic displacement of the amino acid from PLP, leading to changes in overall activity and stability of the α-aminoacrylate intermediate. Data indicate that single amino acid substitutions that yield only subtle changes in structure can produce large differences in reaction rates and overall mechanism.