Kuciel Radosława, Mazurkiewicz Aleksandra, Dudzik Paulina
Chair of Medical Biochemistry, Collegium Medicum, Jagiellonian University, Kraków, Poland.
Acta Biochim Pol. 2007;54(2):371-7. Epub 2007 May 23.
Kinetics of guanidine hydrochloride (GdnHCl)-induced unfolding of human prostatic acid phosphatase (hPAP), a homodimer of 50 kDa subunit molecular mass was investigated with enzyme activity measurements, capacity for binding an external hydrophobic probe, 1-anilinonaphtalene-8-sulfonate (ANS), accessibility of thiols to reaction with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and 2-(4'-maleimidylanilino)naphthalene-6-sulfonate (MIANS) and ability to bind Congo red dye. Kinetic analysis was performed to describe a possible mechanism of hPAP unfolding and dissociation that leads to generation of an inactive monomeric intermediate that resembles, in solution of 1.25 M GdnHCl pH 7.5, at 20 degrees C, in equilibrium, a molten globule state. The reaction of hPAP inactivation in 1.25 M GdnHCl followed first order kinetics with the reaction rate constant 0.0715 +/- 0.0024 min(-1) . The rate constants of similar range were found for the pseudo-first-order reactions of ANS and Congo red binding: 0.0366 +/- 0.0018 min(-1) and 0.0409 +/- 0.0052 min(-1), respectively. Free thiol groups, inaccessible in the native protein, were gradually becoming, with the progress of unfolding, exposed for the reactions with DTNB and MIANS, with the pseudo-first-order reaction rate constants 0.327 +/- 0.014 min(-1) and 0.216 +/- 0.010 min(-1), respectively. The data indicated that in the course of hPAP denaturation exposure of thiol groups to reagents took place faster than the enzyme inactivation and exposure of the protein hydrophobic surface. This suggested the existence of a catalytically active, partially unfolded, but probably dimeric kinetic intermediate in the process of hPAP unfolding. On the other hand, the protein inactivation was accompanied by exposure of a hydrophobic, ANS-binding surface, and with an increased capacity to bind Congo red. Together with previous studies these results suggest that the stability of the catalytically active conformation of the enzyme depends mainly on the dimeric structure of the native hPAP.
通过酶活性测量、与外部疏水探针1-苯胺基萘-8-磺酸盐（ANS）的结合能力、巯基与5,5'-二硫代双（2-硝基苯甲酸）（DTNB）和2-（4'-马来酰亚胺基苯胺基）萘-6-磺酸盐（MIANS）反应的可及性以及与刚果红染料的结合能力，研究了盐酸胍（GdnHCl）诱导的人前列腺酸性磷酸酶（hPAP，一种亚基分子量为50 kDa的同二聚体）的解折叠动力学。进行动力学分析以描述hPAP解折叠和解离的可能机制，该机制导致产生一种无活性的单体中间体，在1.25 M GdnHCl、pH 7.5、20℃的溶液中，处于平衡状态时类似于熔融球状体状态。hPAP在1.25 M GdnHCl中的失活反应遵循一级动力学，反应速率常数为0.0715±0.0024 min⁻¹。对于ANS和刚果红结合的准一级反应，发现了相似范围的速率常数：分别为0.0366±0.0018 min⁻¹和0.0409±0.0052 min⁻¹。天然蛋白质中不可及的游离巯基随着解折叠的进行逐渐暴露，以便与DTNB和MIANS反应，准一级反应速率常数分别为0.327±0.014 min⁻¹和0.216±0.010 min⁻¹。数据表明，在hPAP变性过程中，巯基与试剂的接触比酶失活和蛋白质疏水表面的暴露更快。这表明在hPAP解折叠过程中存在一种催化活性的、部分解折叠但可能是二聚体的动力学中间体。另一方面，蛋白质失活伴随着疏水的、ANS结合表面的暴露以及与刚果红结合能力的增加。与先前的研究一起，这些结果表明该酶催化活性构象的稳定性主要取决于天然hPAP的二聚体结构。
