Chiou Shyh-Ying, Lai Cheng-Yue, Lin Long-Yau, Lin Gialih
Institute of Medicine, Department of Neurosurgery, Chung Shan Medical University, Taichung 402, Taiwan.
BMC Biochem. 2005 Sep 22;6:17. doi: 10.1186/1471-2091-6-17.
Recently there has been increased interest in pancreatic cholesterol esterase due to correlation between enzymatic activity in vivo and absorption of dietary cholesterol. Cholesterol esterase plays a role in digestive lipid absorption in the upper intestinal tract, though its role in cholesterol absorption in particular is controversial. Serine lipases, acetylcholinesterase, butyrylcholinesterase, and cholesterol esterase belong to a large family of proteins called the alpha/beta-hydrolase fold, and they share the same catalytic machinery as serine proteases in that they have an active site serine residue which, with a histidine and an aspartic or glutamic acid, forms a catalytic triad. The aim of this work is to study the stereoselectivity of the acyl chain binding site of the enzyme for four diastereomers of an inhibitor.
Four diastereomers of 2'-N-alpha-methylbenzylcarbamyl-1, 1'-bi-2-naphthol (1) are synthesized from the condensation of R-(+)- or S-(-)-1, 1'-bi-2-naphthanol with R-(+)- or S-(-)-alpha-methylbenzyl isocyanate in the presence of a catalytic amount of pyridine in CH2Cl2. The [alpha]25D values for (1R, alphaR)-1, (1R, alphaS)-1, (1S, alphaR)-1, and (1S, alphaS)-1 are +40, +21, -21, and -41 degrees, respectively. All four diastereomers of inhibitors are characterized as pseudo substrate inhibitors of pancreatic cholesterol esterase. Values of the inhibition constant (Ki), the carbamylation constant (k2), and the bimolecular rate constant (ki) for these four diastereomeric inhibitors are investigated. The inhibitory potencies for these four diastereomers are in the descending order of (1R, alphaR)-1, (1R, alphaS)-1, (1S, alphaR)-1, and (1S, alphaS)-1. The k2 values for these four diastereomers are about the same. The enzyme stereoselectivity for the 1, 1'-bi-2-naphthyl moiety of the inhibitors (R > S, ca. 10 times) is the same as that for 2'-N-butylcarbamyl-1, 1'-bi-2-naphthol (2). The enzyme stereoselectivity for the alpha-methylbenzylcarbamyl moiety of the inhibitors is also R > S (2-3 times) due to the constraints in the acyl binding site.
We are the first to report that the acyl chain binding site of cholesterol esterase shows stereoselectivity for the four diastereomers of 1.
由于体内酶活性与膳食胆固醇吸收之间的相关性，近来人们对胰腺胆固醇酯酶的兴趣有所增加。胆固醇酯酶在上消化道的消化脂质吸收中发挥作用，不过其在胆固醇吸收方面的具体作用存在争议。丝氨酸脂肪酶、乙酰胆碱酯酶、丁酰胆碱酯酶和胆固醇酯酶属于一个名为α/β水解酶折叠的蛋白质大家族，它们与丝氨酸蛋白酶共享相同的催化机制，即都有一个活性位点丝氨酸残基，该残基与一个组氨酸以及一个天冬氨酸或谷氨酸形成催化三联体。本研究的目的是研究该酶的酰基链结合位点对一种抑制剂的四种非对映异构体的立体选择性。
2'-N-α-甲基苄基氨基甲酰基-1,1'-联-2-萘酚（1）的四种非对映异构体由R-(+)-或S-(-)-1,1'-联-2-萘酚与R-(+)-或S-(-)-α-甲基苄基异氰酸酯在二氯甲烷中、催化量的吡啶存在下缩合而成。(1R,αR)-1、(1R,αS)-1、(1S,αR)-1和(1S,αS)-1的[α]25D值分别为+40、+21、-21和-41度。抑制剂的所有四种非对映异构体均被表征为胰腺胆固醇酯酶的假底物抑制剂。研究了这四种非对映体抑制剂的抑制常数（Ki）、氨甲酰化常数（k2）和双分子速率常数（ki）的值。这四种非对映异构体的抑制效力顺序为(1R,αR)-1、(1R,αS)-1、(1S,αR)-1和(1S,αS)-1。这四种非对映异构体的k2值大致相同。酶对抑制剂的1,1'-联-2-萘基部分的立体选择性（R＞S，约10倍）与对2'-N-丁基氨基甲酰基-1,1'-联-2-萘酚（2）的立体选择性相同。由于酰基结合位点的限制，酶对抑制剂的α-甲基苄基氨基甲酰基部分的立体选择性也是R＞S（2 - 3倍）。
我们首次报道胆固醇酯酶的酰基链结合位点对1的四种非对映异构体表现出立体选择性。
