Malver Olaf, Sebastian Mina J, Oppenheimer Norman J
Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, CA 94143-0912, United States.
Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, CA 94143-0912, United States.
DNA Repair (Amst). 2014 Nov;23:95-100. doi: 10.1016/j.dnarep.2014.09.005. Epub 2014 Oct 3.
A new, acyclic NAD-analog, acycloNAD(+) has been synthesized where the nicotinamide ribosyl moiety has been replaced by the nicotinamide (2-hydroxyethoxy)methyl moiety. The chemical properties of this analog are comparable to those of β-NAD(+) with a redox potential of -324mV and a 341nm λmax for the reduced form. Both yeast alcohol dehydrogenase (YADH) and horse liver alcohol dehydrogenase (HLADH) catalyze the reduction of acycloNAD(+) by primary alcohols. With HLADH 1-butanol has the highest Vmax at 49% that of β-NAD(+). The primary deuterium kinetic isotope effect is greater than 3 indicating a significant contribution to the rate limiting step from cleavage of the carbon-hydrogen bond. The stereochemistry of the hydride transfer in the oxidation of stereospecifically deuterium labeled n-butanol is identical to that for the reaction with β-NAD(+). In contrast to the activity toward primary alcohols there is no detectable reduction of acycloNAD(+) by secondary alcohols with HLADH although these alcohols serve as competitive inhibitors. The net effect is that acycloNAD(+) has converted horse liver ADH from a broad spectrum alcohol dehydrogenase, capable of utilizing either primary or secondary alcohols, into an exclusively primary alcohol dehydrogenase. This is the first example of an NAD analog that alters the substrate specificity of a dehydrogenase and, like site-directed mutagenesis of proteins, establishes that modifications of the coenzyme distance from the active site can be used to alter enzyme function and substrate specificity. These and other results, including the activity with α-NADH, clearly demonstrate the promiscuity of the binding interactions between dehydrogenases and the riboside phosphate of the nicotinamide moiety, thus greatly expanding the possibilities for the design of analogs and inhibitors of specific dehydrogenases.
一种新的、无环的烟酰胺腺嘌呤二核苷酸类似物——无环烟酰胺腺嘌呤二核苷酸（acycloNAD(+)）已被合成，其中烟酰胺核糖基部分被烟酰胺（2-羟基乙氧基）甲基部分所取代。该类似物的化学性质与β-NAD(+)相当，其氧化还原电位为-324mV，还原形式的最大吸收波长为341nm。酵母乙醇脱氢酶（YADH）和马肝乙醇脱氢酶（HLADH）都能催化伯醇还原acycloNAD(+)。对于HLADH，1-丁醇的最大反应速度（Vmax）最高，为β-NAD(+)的49%。初级氘动力学同位素效应大于3，表明碳氢键的断裂对限速步骤有显著贡献。立体特异性氘标记正丁醇氧化过程中氢化物转移的立体化学与与β-NAD(+)反应的立体化学相同。与对伯醇的活性相反，仲醇与HLADH不能检测到acycloNAD(+)的还原，尽管这些醇可作为竞争性抑制剂。总体效果是acycloNAD(+)已将马肝乙醇脱氢酶从一种能够利用伯醇或仲醇的广谱乙醇脱氢酶转变为一种专一性的伯醇脱氢酶。这是烟酰胺腺嘌呤二核苷酸类似物改变脱氢酶底物特异性的首个例子，并且与蛋白质的定点诱变一样，表明改变辅酶与活性位点的距离可用于改变酶的功能和底物特异性。这些结果以及其他结果，包括与α-NADH的活性，清楚地证明了脱氢酶与烟酰胺部分的核糖磷酸之间结合相互作用的混杂性，从而极大地扩展了设计特定脱氢酶类似物和抑制剂的可能性。
