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Probing the enzyme catalytic mechanism by nuclear magnetic resonance - A case study of a serine protease

机译:通过核磁共振探究酶催化机理-以丝氨酸蛋白酶为例

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Serine proteases are among the most studied enzymes for their role as model enzymes for studying the enzyme catalytic mechanism and medical interest in their inhibition. We have applied NMR methods to determine the structure, dynamics, and catalytic mechanism of a serine protease, E. coli thioesterase/protease I (TEP-I). In this article we review the results of our efforts. We showed that TEP-I is an alpha/beta/alpha type SNGH-hydrolase with Ser1(0), Asp(154) and His(157) as the catalytic triad residues. In free TEP-1, His(157) was found to form a strong hydrogen bond to Asp(154), but not to Ser(10)-(OH)-H-gamma. Modelfree analysis of N-15-T-1, N-15-T-2 and H-1-N-15 NOE data revealed that TEP-I is a rigid protein with a flexible catalytic binding pocket. Slow motion involving segments around the catalytic site was detected. The formation of Michaelis complex (MC) between TEP-I and a transition state analogue, diethyl p-nitrophenyl phosphate (DENP), and its subsequent conversion to the tetrahedral complex (TC) follow a two-step process, a fast formation of MC followed by a slow conversion to TC. In both steps residues perturbed were confined mainly to four conserved segments comprising the active site. Comparable magnitudes of chemical shift perturbations were detected in both steps. From the large chemical shift perturbation upon conversion from MC to TC we proposed that the amide protons of Ser(10) and Gly(44) serve as the oxyanion-hole proton donors to stabilize the tetrahedral adduct. The pattern of residues perturbed in both steps suggests a sequential, stepwise structural change upon binding of DENP.
机译:丝氨酸蛋白酶由于其作为模型酶的作用而被研究最多的酶,用于研究酶的催化机理和医学上对其抑制的兴趣。我们已应用NMR方法确定丝氨酸蛋白酶大肠杆菌硫酯酶/蛋白酶I(TEP-1)的结构,动力学和催化机理。在本文中,我们回顾了我们的努力结果。我们表明,TEP-1是具有Ser1(0),Asp(154)和His(157)作为催化三联体残基的alpha / beta / alpha型SNGH水解酶。在游离TEP-1中,发现His(157)与Asp(154)形成强氢键,但与Ser(10)-(OH)-H-γ形成强氢键。 N-15-T-1,N-15-T-2和H-1-N-15 NOE数据的无模型分析表明,TEP-1是一种具有柔性催化结合袋的刚性蛋白质。慢动作检测到催化部位周围的片段。 TEP-1和过渡态类似物对二苯基磷酸对硝基苯酯(DENP)之间的Michaelis配合物(MC)的形成以及随后的转化为四面体配合物(TC)的过程分为两步,即快速形成MC然后缓慢转换为TC。在这两个步骤中,被干扰的残基主要限制在包含活性位点的四个保守区段上。在两个步骤中都检测到了可比较的化学位移扰动幅度。从MC转换为TC时发生的大化学位移扰动,我们提出Ser(10)和Gly(44)的酰胺质子充当氧阴离子孔质子供体,以稳定四面体加合物。在两个步骤中受到干扰的残基模式表明,DENP结合后会发生连续的,逐步的结构变化。

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