Singh Vipender, Lee Jeffrey E, Núñez Sara, Howell P Lynne, Schramm Vern L
Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA.
Biochemistry. 2005 Sep 6;44(35):11647-59. doi: 10.1021/bi050863a.
Methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) catalyzes reactions linked to polyamine metabolism, quorum sensing pathways, methylation reactions, and adenine salvage. It is a candidate target for antimicrobial drug design. Kinetic isotope effects (KIEs) were measured on the MTAN-catalyzed hydrolysis of 5'-methylthioadenosine (MTA) to determine the transition state structure. KIEs measured at pH 7.5 were near unity due to the large forward commitment to catalysis. Intrinsic KIEs were expressed by increasing the pH to 8.5. Intrinsic KIEs from MTAs labeled at 1'-(3)H, 1'-(14)C, 2'-(3)H, 4'-(3)H, 5'-(3)H, 9-(15)N, and Me-(3)H(3) were 1.160 +/- 0.004, 1.004 +/- 0.003, 1.044 +/- 0.004, 1.015 +/- 0.002, 1.010 +/- 0.002, 1.018 +/- 0.006, and 1.051 +/- 0.002, respectively. The large 1'-(3)H and small 1'-(14)C KIEs indicate that the Escherichia coli MTAN reaction undergoes a dissociative (D(N)A(N)) (S(N)1) mechanism with little involvement of the leaving group or participation of the attacking nucleophile at the transition state, causing the transition state to have significant ribooxacarbenium ion character. A transition state constrained to match the intrinsic KIEs was located with density functional theory [B3LYP/6-31G(d,p)]. The leaving group (N9) is predicted to be 3.0 A from the anomeric carbon. The small beta-secondary 2'-(3)H KIE of 1.044 corresponds to a modest 3'-endo conformation for ribose and a H1'-C1'-C2'-H2' dihedral angle of 53 degrees at the transition state. Natural bond orbital analysis of the substrate and the transition state suggests that the 4'-(3)H KIE is due to hyperconjugation between the lone pair (n(p)) of O3' and the antibonding (sigma) orbital of the C4'-H4' group, and the methyl-(3)H(3) KIE is due to hyperconjugation between the n(p) of sulfur and the sigma of methyl C-H bonds. Transition state analogues that resemble this transition state structure are powerful inhibitors, and their molecular electrostatic potential maps closely resemble that of the transition state.
甲硫基腺苷/S-腺苷高半胱氨酸核苷酶（MTAN）催化与多胺代谢、群体感应途径、甲基化反应和腺嘌呤补救相关的反应。它是抗菌药物设计的候选靶点。通过测量MTAN催化的5'-甲硫基腺苷（MTA）水解反应的动力学同位素效应（KIEs）来确定过渡态结构。在pH 7.5时测得的KIEs接近1，这是由于催化过程中较大的正向反应倾向。通过将pH提高到8.5来表达本征KIEs。标记有1'-(3)H、1'-(14)C、2'-(3)H、4'-(3)H、5'-(3)H、9-(15)N和Me-(3)H(3)的MTA的本征KIEs分别为1.160±0.004、1.004±0.003、1.044±0.004、1.015±0.002、1.010±0.002、1.018±0.006和1.051±0.002。较大的1'-(3)H KIE和较小的1'-(14)C KIE表明，大肠杆菌MTAN反应经历解离（D(N)A(N)）（S(N)1）机制，在过渡态时离去基团参与很少，亲核进攻试剂参与也很少，导致过渡态具有显著的核糖氧碳鎓离子特征。利用密度泛函理论[B3LYP/6-3（d,p）]确定了与本征KIEs相匹配的过渡态。预测离去基团（N9）与异头碳的距离为3.0 Å。较小的β-二级2'-(3)H KIE为1.044，对应于核糖在过渡态时适度的3'-内型构象以及H[1']-C[1']-C[2']-H[2']二面角为５3°。对底物和过渡态的自然键轨道分析表明，4'-(3)H KIE是由于O3'的孤对电子（n(p)）与C4'-H4'基团的反键（σ）轨道之间的超共轭作用，而甲基-(3)H(3) KIE是由于硫的n(p)与甲基C-H键的σ之间的超共轭作用。类似于这种过渡态结构的过渡态类似物是强力抑制剂，它们的分子静电势图与过渡态的非常相似。
