Chin J K, Klinman J P
Departments of Chemistry and Molecular and Cell Biology, University of California, Berkeley, California 94720, USA.
Biochemistry. 2000 Feb 15;39(6):1278-84. doi: 10.1021/bi9920331.
A tunneling contribution to hydride transfer has been demonstrated previously in the oxidation of benzyl alcohol catalyzed by an active-site mutant (F93W) of horse liver alcohol dehydrogenase (LADH) [Bahnson, B. J., et al. (1993) Biochemistry 32, 5503-5507]. Mutation of a residue that lies directly behind the nicotinamide ring of the bound cofactor has further shown that side-chain bulk can contribute to catalytic efficiency and tunneling in a correlated fashion [Bahnson, B. J., et al. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 12797-12802]. Second site mutations of F93W have now been made at positions more remote from the active site. In particular, we have focused on an isoleucine residue that interacts with the adenine moiety of the NAD(+) cofactor, 20 A from the nicotinamide ring. Replacement of this remote residue with glycine (F93W:I224G), alanine (F93W:I224A), valine (F93W:I224V), and leucine (F93W:I224L) is concluded to destabilize the binding of NAD(+). All double mutants exhibited a K(M) for NAD(+) that is 2-25 times higher than that for the F93W enzyme. However, neither the catalytic efficiency for turnover of benzyl alcohol [k(cat)/K(M(benzyl alcohol))] nor the relationship between the secondary k(H)/k(T) and k(D)/k(T) isotope effects for benzyl alcohol oxidation was significantly affected. The lack of differences observed in the isotope effects indicates that these mutations have little effect on the extent of hydrogen tunneling in the reaction. The complete removal of the side chain at position 224 in the F93W:I224G enzyme resulted in a less than 5% decrease in the ratio of the secondary isotope effects, maintaining the ratio above the semiclassical limit for the indication of tunneling in the reaction. By contrast, K(i) for NAD(+) increased 60-fold for this mutant. The results obtained with F93W:I224G are consistent with remote interactions that affect the association and binding of cofactor in a reactive conformation. However, once this conformation is achieved, hydride transfer and its tunneling component proceed as with the single F93W mutant enzyme, uninfluenced by the remote mutation. Replacement of other side chains, with alpha-carbon positions from about 8 to over 20 A from the C4 position of the nicotinamide ring, demonstrated a similar insensitivity of k(cat)/K(M(benzyl alcohol)) to protein modification. Comparison to earlier studies with active-site mutants of LADH implicates a role for proximal, but not distal, side chains in the modulation of hydrogen tunneling for this enzyme.
先前已证明，在马肝醇脱氢酶（LADH）的活性位点突变体（F93W）催化苯甲醇氧化反应中，存在氢负离子转移的隧穿贡献[Bahnson, B. J., 等人（1993年）《生物化学》32卷，5503 - 5507页]。结合辅因子烟酰胺环正后方一个残基的突变进一步表明，侧链体积能以相关方式影响催化效率和隧穿[Bahnson, B. J., 等人（1997年）《美国国家科学院院刊》94卷，12797 - 12802页]。现在已在距离活性位点更远的位置对F93W进行了第二位点突变。特别地，我们聚焦于一个与NAD(+)辅因子的腺嘌呤部分相互作用、距离烟酰胺环20 Å的异亮氨酸残基。用甘氨酸（F93W:I224G）、丙氨酸（F93W:I224A）、缬氨酸（F93W:I224V）和亮氨酸（F93W:I224L）取代这个远端残基，结果表明会破坏NAD(+)的结合。所有双突变体对NAD(+)的K(M)值比对F93W酶高2 - 25倍。然而，苯甲醇周转的催化效率[k(cat)/K(M(苯甲醇))]以及苯甲醇氧化反应中二级k(H)/k(T)与k(D)/k(T)同位素效应之间的关系均未受到显著影响。在同位素效应方面未观察到差异，这表明这些突变对反应中氢隧穿的程度影响很小。F93W:I224G酶中224位侧链的完全去除导致二级同位素效应的比值下降不到5%，该比值仍高于反应中表明隧穿的半经典极限。相比之下，该突变体对NAD(+)的K(i)值增加了60倍。F93W:I224G的结果与影响辅因子以反应性构象缔合和结合的远程相互作用一致。然而，一旦达到这种构象，氢负离子转移及其隧穿组分就如同单个F93W突变酶一样进行，不受远程突变的影响。用距离烟酰胺环C4位置约8 Å至超过20 Å的α - 碳原子位置的其他侧链进行取代，结果表明k(cat)/K(M(苯甲醇))对蛋白质修饰同样不敏感。与先前对LADH活性位点突变体的研究相比，表明近端而非远端侧链在调节该酶的氢隧穿中起作用。
