Tanner J J, Hecht R M, Krause K L
Department of Biochemical and Biohysical Sciences, University of Houston, Houston, Texas 77204-5934, USA.
Biochemistry. 1996 Feb 27;35(8):2597-609. doi: 10.1021/bi951988q.
The crystal structure of holo D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the extreme thermophile Thermus aquaticus has been solved at 2.5 Angstroms resolution. To study the determinants of thermostability, we compare our structure to four other GAPDHs. Salt links, hydrogen bonds, buried surface area, packing density, surface to volume ratio, and stabilization of alpha-helices and beta-turns are analyzed. We find a strong correlation between thermostability and the number of hydrogen bonds between charged side chains and neutral partners. These charged-neutral hydrogen bonds provide electrostatic stabilization without the heavy desolvation penalty of salt links. The stability of thermophilic GAPDHs is also correlated with the number of intrasubunit salt links and total hydrogen bonds. Charged residues, therefore, play a dual role in stabilization by participating not only in salt links but also in hydrogen bonds with a neutral partner. Hydrophobic effects allow for discrimination between thermophiles and psychrophiles, but not within the GAPDH thermophiles. There is, however, an association between thermostability and decreasing enzyme surface to volume ratio. Finally, we describe several interactions present in both our GAPDH and a hyperthermophilic GAPDH that are absent in the less thermostable GAPDHs. These include a four-residue salt link network, a hydrogen bond near the active site, an intersubunit salt link, and several buried Ile residues.
嗜热栖热菌全酶D -甘油醛- 3 -磷酸脱氢酶（GAPDH）的晶体结构已在2.5埃分辨率下解析出来。为了研究热稳定性的决定因素，我们将我们解析的结构与其他四种GAPDH进行比较。分析了盐桥、氢键、埋藏表面积、堆积密度、表面积与体积比以及α螺旋和β转角的稳定性。我们发现热稳定性与带电侧链和中性配体之间氢键的数量之间存在很强的相关性。这些带电-中性氢键提供了静电稳定作用，而没有盐桥那种严重的去溶剂化代价。嗜热GAPDH的稳定性也与亚基内盐桥的数量和总氢键数量相关。因此，带电残基在稳定化过程中发挥双重作用，不仅参与盐桥，还与中性配体形成氢键。疏水作用使得嗜热菌和嗜冷菌之间存在差异，但在GAPDH嗜热菌内部不存在这种差异。然而，热稳定性与酶表面积与体积比的降低之间存在关联。最后，我们描述了在我们解析的GAPDH和一种超嗜热GAPDH中都存在的几种相互作用，而在热稳定性较低的GAPDH中不存在。这些相互作用包括一个四残基盐桥网络、活性位点附近的一个氢键、一个亚基间盐桥以及几个埋藏的异亮氨酸残基。
