Comparative Kinetics of Cofactor Association and Dissociation for the Human and Trypanosomal S-Adenosylhomocysteine Hydrolases. 1. Basic Features of the Association and Dissociation Processes
详细信息 查看全文
- 作者：Qing-Shan Li ; Sumin Cai ; Ronald T. Borchardt ; Jianwen Fang ; Krzysztof Kuczera ; C. Russell Middaugh ; Richard L. Schowen
- 刊名：Biochemistry
- 出版年：2007
- 出版时间：May 15, 2007
- 年：2007
- 卷：46
- 期：19
- 页码：5798 - 5809
- 全文大小：155K
- 年卷期：v.46,no.19(May 15, 2007)
- ISSN：1520-4995
文摘
The S-adenosyl-L-homocysteine (AdoHcy) hydrolases catalyze the reversible conversion ofAdoHcy to adenosine and homocysteine, making use of a catalytic cycle in which a tightly bound NAD+oxidizes the 3'-hydroxyl group of the substrate at the beginning of the cycle, activating the 4'-CH bondfor elimination of homocysteine, followed by Michael addition of water to the resulting intermediate anda final reduction by the tightly bound NADH to give adenosine. The equilibrium and kinetic propertiesof the association and dissociation of the cofactor NAD+ from the enzymes of Homo sapiens (Hs-SAHH)and Trypanosoma cruzi (Tc-SAHH) are qualitatively similar but quantitatively distinct. Both enzymesbind NAD+ in a complex scheme. The four active sites of the homotetrameric apoenzyme appear todivide into two numerically equal classes of active sites. One class of sites binds cofactor weakly andgenerates full activity very rapidly (in less than 1 min). The other class binds cofactor more strongly butgenerates activity only slowly (>30 min). In the case of Tc-SAHH, the final affinity for NAD+ is roughlymicromolar and this affinity persists as the equilibrium affinity. In the case of Hs-SAHH, the slow-binding phase terminates in micromolar affinity also, but over a period of hours, the dissociation rateconstant decreases until the final equilibrium affinity is in the nanomolar range. The slow binding ofNAD+ by both enzymes exhibits saturation kinetics with respect to the cofactor concentration; however,binding to Hs-SAHH has a maximum rate constant around 0.06 s-1, while the rate constant for bindingto Tc-SAHH levels out at 0.006 s-1. In contrast to the complex kinetics of association, both enzymesundergo dissociation of NAD+ from all four sites in a single first-order reaction. The equilibrium affinitiesof both Hs-SAHH and Tc-SAHH for NADH are in the nanomolar range. The dissociation rate constantsand the slow-binding association rate constants for NAD+ show a complex temperature dependence withboth enzymes; however, the cofactor always dissociates more rapidly from Tc-SAHH than from Hs-SAHH, the ratio being around 80-fold at 37 
C, and the cofactor binds more rapidly to Hs-SAHH thanto Tc-SAHH above ~16 C. These features present an opening for selective inhibition of Tc-SAHH overHs-SAHH, demonstrated with the thioamide analogues of NAD+ and NADH. Both analogues bind toHs-SAHH with ~40 nM affinities but much more weakly to Tc-SAHH (0.6-15 
M). Nevertheless, bothanalogues inactivated Tc-SAHH 60% (NAD+ analogue) or 100% (NADH analogue) within 30 min, whilethe degree of inhibition of Hs-SAHH approached 30% only after 12 h. The rate of loss of activity is equalto the rate of dissociation of the cofactor and thus 80-fold faster at 37 C for Tc-SAHH.
C, and the cofactor binds more rapidly to Hs-SAHH thanto Tc-SAHH above ~16 C. These features present an opening for selective inhibition of Tc-SAHH overHs-SAHH, demonstrated with the thioamide analogues of NAD+ and NADH. Both analogues bind toHs-SAHH with ~40 nM affinities but much more weakly to Tc-SAHH (0.6-15 M). Nevertheless, bothanalogues inactivated Tc-SAHH 60% (NAD+ analogue) or 100% (NADH analogue) within 30 min, whilethe degree of inhibition of Hs-SAHH approached 30% only after 12 h. The rate of loss of activity is equalto the rate of dissociation of the cofactor and thus 80-fold faster at 37 C for Tc-SAHH.