Ogata H, Fukuda T, Yamamoto K, Funakoshi J, Takada K, Yasue N, Fujisaki S, Kajigaeshi S
Division of Chemistry and Biochemistry, The School of Allied Health Sciences, Yamaguchi University, Japan.
Biochim Biophys Acta. 1992 Feb 26;1119(2):123-6. doi: 10.1016/0167-4838(92)90381-m.
Alkyl glycolamido phosphoric esters (P-O-CH2-CO-NH-(CH2)n-CH3) and alkyl monoglycolate phosphoric esters (P-O-CH2-CO-O-(CH2)n-CH3), which are analogs of the aldolase substrate fructose-1-phosphate, were synthesized and use for probing the active site of rabbit muscle aldolase. The inhibition constants (Ki) were affected by the length of the alkyl groups of these compounds and a maximum value of Ki was observed between the number of methylene groups 2 and 4, depending on the type of compound. In the previous investigation, N-(omega-hydroxyalkyl)-glycolamido bisphosphoric esters (P-O-CH2-CO-NH-(CH2)n-O-P) and alkanediol monoglyclolate bisphosphoric esters (P-O-CH2-CO-O-(CH2)n-O-P) have a minimum Ki value between the number of methylene groups 1 and 4. The difference spectra of aldolase caused by binding of alkyl glycoamido phosphoric esters or alkyl monophosphates resembled that of their analogous bisphosphoric esters, but the intensity of absorbance was smaller than that of the bisphosphoric ester analogs. These results suggest that rabbit muscle aldolase has two binding sites for the phosphate groups on the entrance end of the active site cavity, the singly wound beta-barrel of the parallel alpha/beta class structure. The distance between the phosphate binding site Lys-107 in the beta-sheet structure (c) and Arg-148 in the beta-sheet structure (d) may possibly be expanded or contracted by the forms of the bending structure of the biphosphate compounds. Also, the change of distance between the beta-sheet structure (c) and (d) containing Trp-147, may have an effect on the environment of the tryptophan and cause a change of the absorbance of aldolase especially at 295-299 nm. On the other hand, the synthetic monophosphate compounds bind at only one of the two phosphate binding sites and have very little effect on the absorbance of Trp-147, in a similar manner as orthophosphate. The alkyl groups of monophosphate may be repelled by the ionic amino acid side chains, Asp-33, Lys-146, Glu-187 and/or Lys-229 in the middle of the active site cavity. However, the end of the long alkyl group of some monophosphates may possibly contact the hydrophobic bottom of the active site cavity without effect on the environment of Trp-147.
合成了醛缩酶底物1-磷酸果糖的类似物烷基甘醇酰胺磷酸酯（P-O-CH₂-CO-NH-(CH₂)ₙ-CH₃）和烷基单乙醇酸磷酸酯（P-O-CH₂-CO-O-(CH₂)ₙ-CH₃），并用于探测兔肌肉醛缩酶的活性位点。这些化合物的抑制常数（Ki）受烷基长度的影响，根据化合物类型，在亚甲基数目为2至4时观察到Ki的最大值。在先前的研究中，N-（ω-羟烷基）-甘醇酰胺双磷酸酯（P-O-CH₂-CO-NH-(CH₂)ₙ-O-P）和链烷二醇单乙醇酸双磷酸酯（P-O-CH₂-CO-O-(CH₂)ₙ-O-P）在亚甲基数目为1至4时具有最小的Ki值。由烷基甘醇酰胺磷酸酯或烷基单磷酸酯结合引起的醛缩酶的差光谱类似于它们相应的双磷酸酯的差光谱，但吸光度强度小于双磷酸酯类似物。这些结果表明，兔肌肉醛缩酶在活性位点腔入口端有两个磷酸基团结合位点，该活性位点腔是平行α/β类结构的单股β-桶。β-折叠结构（c）中的磷酸结合位点Lys-107与β-折叠结构（d）中的Arg-148之间的距离可能会因双磷酸化合物弯曲结构的形式而扩大或收缩。此外，含有Trp-147的β-折叠结构（c）和（d）之间距离的变化，可能会影响色氨酸的环境，并导致醛缩酶吸光度的变化，尤其是在295 - 299 nm处。另一方面，合成的单磷酸化合物仅结合在两个磷酸结合位点中的一个，并且对Trp-147的吸光度影响很小，与正磷酸的情况类似。单磷酸酯的烷基可能被活性位点腔中部的离子型氨基酸侧链Asp-33、Lys-146、Glu-187和/或Lys-229排斥。然而，一些单磷酸酯的长烷基末端可能会接触活性位点腔的疏水底部，而不影响Trp-147的环境。
