Large-Scale Domain Movements and Hydration Structure Changes in the Active-Site Cleft of Unligated Glutamate Dehydrogenase from Thermococcus profundus Studied by Cryogenic X-ray Crystal Structu

详细信息    查看全文

• 作者：Masayoshi Nakasako ; Testuro Fujisawa ; Shin-ichi Adachi ; Toshiaki Kudo ; and Sadaharu Higuchi
• 刊名：Biochemistry
• 出版年：2001
• 出版时间：March 13, 2001
• 年：2001
• 卷：40
• 期：10
• 页码：3069 - 3079
• 全文大小：2369K
• 年卷期：v.40,no.10(March 13, 2001)
• ISSN：1520-4995

文摘

Here we describe the large-scale domain movements and hydration structure changes in theactive-site cleft of unligated glutamate dehydrogenase. Glutamate dehydrogenase from Thermococcusprofundus is composed of six identical subunits of M_r 46K, each with two distinct domains of roughlyequal size separated by a large active-site cleft. The enzyme in the unligated state was crystallized so thatone hexamer occupied a crystallographic asymmetric unit, and the crystal structure of the hexamer wassolved and refined at a resolution of 2.25 Å with a crystallographic R-factor of 0.190. In that structure,the six subunits displayed significant conformational variations with respect to the orientations of the twodomains. The variation was most likely explained as a hinge-bending motion caused by small changes inthe main chain torsion angle of the residue composing a loop connecting the two domains. Small-angleX-ray scattering profiles both at 293 and 338 K suggested that the apparent molecular size of the hexamerwas slightly larger in solution than in the crystalline state. These results led us to the conclusion that (i)the spontaneous domain motion was the property of the enzyme in solution, (ii) the domain motion wastrapped in the crystallization process through different modes of crystal contacts, and (iii) the magnitudeof the motion in solution was greater than that observed in the crystal structure. The present cryogenicdiffraction experiment enabled us to identify 1931 hydration water molecules around the hexamer. Thehydration structures around the subunits exhibited significant changes in accord with the degree of thedomain movement. In particular, the hydration water molecules in the active-site cleft were rearrangedmarkedly through migrations between specific hydration sites in coupling strongly with the domainmovement. We discussed the cooperative dynamics between the domain motion and the hydration structurechanges in the active site of the enzyme. The present study provides the first example of a visualizedhydration structure varying transiently with the dynamic movements of enzymes and may form a newconcept of the dynamics of multidomain enzymes in solution.