Substrate Binding Stabilizes S-Adenosylhomocysteine Hydrolase in a Closed Conformation

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• 作者：Dan Yin ; Xiaoda Yang ; Yongbo Hu ; Krysztof Kuczera ; Richard L. Schowen ; Ronald T. Borchardt ; and Thomas C. Squier
• 刊名：Biochemistry
• 出版年：2000
• 出版时间：August 15, 2000
• 年：2000
• 卷：39
• 期：32
• 页码：9811 - 9818
• 全文大小：94K
• 年卷期：v.39,no.32(August 15, 2000)
• ISSN：1520-4995

文摘

Comparison of crystal structures of S-adenosylhomocysteine (AdoHcy) hydrolase in thesubstrate-free, NAD⁺ form [Hu, Y., Komoto, J., Huang, Y., Gomi, T., Ogawa, H., Takata, Y., Fujioka,M., and Takusagawa, F. (1999) Biochemistry 38, 8323-8333] and a substrate-bound, NADH form [Turner,M. A., Yuan, C.-S., Borchardt, R. T., Hershfield, M. S., Smith, G. D., and Howell, P. L. (1998) Nat.Struct. Biol. 5, 369-376] indicates large differences in the spatial arrangement of the catalytic and NAD⁺binding domains. The substrate-free, NAD⁺ form exists in an "open" form with respect to catalytic andNAD⁺ binding domains, whereas the substrate-bound, NADH form exists in a closed form with respectto those domains. To address whether domain closure is induced by substrate binding or its subsequentoxidation, we have measured the rotational dynamics of spectroscopic probes covalently bound to Cys¹¹³and Cys⁴²¹ within the catalytic and carboxyl-terminal domains. An independent domain motion is associatedwith the catalytic domain prior to substrate binding, suggesting the presence of a flexible hinge elementbetween the catalytic and NAD⁺ binding domains. Following binding of substrates (i.e., adenosine orneplanocin A) or a nonsubstrate (i.e., 3'-deoxyadenosine), the independent domain motion associatedwith the catalytic domain is essentially abolished. Likewise, there is a substantial decrease in the averagehydrodynamic volume of the protein that is consistent with a reduction in the overall dimensions of thehomotetrameric enzyme following substrate binding and oxidation observed in earlier crystallographicstudies. Thus, the catalytic and NAD⁺ binding domains are stabilized to form a closed active site throughinteractions with the substrate prior to substrate oxidation.