Kinetic and structural analysis of human steroid 5beta-reductase (AKR1D1)

描述(申请人提供)：人类固醇-52-还原酶(Aldo-keto Reductase 1d1，AKR1D1)是胆汁酸生物合成和类固醇激素代谢中最关键的酶之一。52-还原赋予胆汁酸类似洗涤剂的特性来乳化脂肪和脂溶维生素。AKR1D1还可以使雄激素、孕激素和糖皮质激素失活，并启动类固醇激素清除。AKR1D1的缺陷会导致新生儿肝炎和胆汁淤积，这可能是致命的。AKR1D1在性功能和生殖功能中的确切作用仍在调查中。AKR1D1催化类固醇-52还原的机制尚未完全阐明。这项建议的目的是建立AKR1D1的动力学和化学机制，并研究自然突变P133R导致疾病的分子基础。在本项目的第一个目标中，将阐明AKR1D1的完整动力学机制。将采用稳态和暂态动力学测量相结合的方法来确定宏观速率常数，并确定52-还原所涉及的速率决定步骤。将利用初级和溶剂动力学同位素效应来研究双键还原的化学机理。第二个目标将集中在P133R突变体的动力学分析和X射线晶体结构测定上。为了抑制P133R突变体的活性，提出了一种缓慢的类固醇产物释放过程。P133R突变体的宏观速率常数将被阐明，并与野生型AKR1D1的宏观速率常数进行比较以验证假设。突变体的晶体结构将阐明含有残基P133的环中的结构变化，这些变化可能会影响底物结合和催化。

DESCRIPTION (provided by applicant): Human steroid-52-reductase (aldo-keto reductase 1D1, AKR1D1) is one of the most critical enzymes in bile acid biosynthesis and steroid hormone metabolism. 52-Reduction grants bile acids the detergent-like characteristics to emulsify fats and fat soluble vitamins. AKR1D1 also inactivates androgens, progestins and glucocorticoids and initiates steroid hormone clearance. Deficiency in AKR1D1 induces neonatal hepatitis and cholestasis that can be fatal. The exact role of AKR1D1 in sexual and reproductive functions is still under investigation. The mechanism of steroid-52-reduction catalyzed by AKR1D1 has not been rigorously elucidated. The objective of this proposal is to establish the kinetic and chemical mechanisms for AKR1D1 and investigate the molecular basis by which a natural mutation P133R leads to disease. In the first aim of this project, the complete kinetic mechanism for AKR1D1 will be elucidated. A combination of steady-state and transient-state kinetic measurements will be employed to determine macroscopic rate constants and identify the rate-determining step involved in 52-reduction. Primary and solvent kinetic isotope effects will be utilized to address the chemical mechanism of the double bond reduction. The second aim will focus on the kinetic analysis and X-ray crystal structure determination of the P133R mutant. A slow steroid product release process was proposed to depress P133R mutant activity. The macroscopic rate constants of the P133R mutant will be elucidated and compared to that of the wild type AKR1D1 to validate the hypothesis. The crystal structure of the mutant will illuminate structural changes in the loop containing residue P133 that may affect substrate binding and catalysis.