Schmidt A, Gübitz G M, Kratky C
Institut für Physikalische Chemie, Abteilung für Strukturbiologie, Karl-Franzens Universität Graz, Austria.
Biochemistry. 1999 Feb 23;38(8):2403-12. doi: 10.1021/bi982108l.
Following a recent low-temperature crystal structure analysis of the native xylanase from Penicillium simplicissimum [Schmidt et al. (1998) Protein Sci. 7, 2081-2088], where an array of glycerol molecules, diffused into the crystal during soaking in a cryoprotectant, was observed within the active-site cleft, we utilized monomeric xylose as well as a variety of linear (Xn, n = 2 to 5) and branched xylooligomers at high concentrations (typically 20% w/v) as cryoprotectant for low-temperature crystallographic experiments. Binding of the glycosidic moiety (or its hydrolysis products) to the enzyme's active-site cleft was observed after as little as 30 s soaking of a native enzyme crystal. The use of a substrate or substrate analogue as cryoprotectant therefore suggests itself as a simple and widely applicable alternative to the use of crystallographic flow-cells for substrate-saturation experiments. Short-chain xylooligomers, i.e., xylobiose (X2) and xylotriose (X3), were found to bind to the active-site cleft with its reducing end hydrogen-bonded to the catalytic acid-base catalyst Glu132. Xylotetraose (X4) and -pentaose (X5) had apparently been cleaved during the soaking time into a xylotriose plus a monomeric (X4) or dimeric (X5) sugar. While the trimeric hydrolysis product was always found to bind in the same way as xylotriose, the monomer or dimer yielded only weak and diffuse electron density within the xylan-binding cleft, at the opposite side of the active center. This suggests that the two catalytic residues divide the binding cleft into a "substrate recognition area" (from the active site toward the nonreducing end of a bound xylan chain), with strong and specific xylan binding and a "product release area" with considerably weaker and less specific binding. The size of the substrate recognition area (3-4 subsites for sugar rings) explains enzyme kinetic data, according to which short oligomers (X2 and X3) bind to the enzyme without being hydrolyzed.
最近对简单青霉天然木聚糖酶进行了低温晶体结构分析[施密特等人（1998年），《蛋白质科学》7，2081 - 2088]，在该分析中，于低温保护剂浸泡过程中扩散到晶体中的甘油分子阵列在活性位点裂隙中被观察到。之后，我们使用单体木糖以及各种高浓度（通常为20% w/v）的线性（Xn，n = 2至5）和分支木寡糖作为低温晶体学实验的低温保护剂。在天然酶晶体浸泡仅30秒后，就观察到糖苷部分（或其水解产物）与酶的活性位点裂隙结合。因此，使用底物或底物类似物作为低温保护剂，对于底物饱和实验而言，是一种简单且广泛适用的替代使用晶体学流通池的方法。发现短链木寡糖，即木二糖（X2）和木三糖（X3），以其还原端与催化酸碱催化剂Glu132形成氢键的方式结合到活性位点裂隙。木四糖（X4）和木五糖（X5）在浸泡期间显然已被切割成木三糖加单体（X4）或二聚体（X5）糖。虽然总是发现三聚体水解产物以与木三糖相同的方式结合，但单体或二聚体在活性中心相对侧的木聚糖结合裂隙内仅产生微弱且弥散的电子密度。这表明两个催化残基将结合裂隙分为一个“底物识别区域”（从活性位点朝向结合木聚糖链的非还原端），具有强且特异性的木聚糖结合，以及一个“产物释放区域”，其结合明显较弱且特异性较低。底物识别区域（糖环的3 - 4个亚位点）的大小解释了酶动力学数据，据此短寡糖（X2和X3）结合到酶上而不被水解。
