Department of Biochemistry, University of Texas Health Science Center, San Antonio, TX 78229, USA.
Arch Biochem Biophys. 2012 Dec 1;528(1):45-9. doi: 10.1016/j.abb.2012.08.007. Epub 2012 Aug 30.
Polyamine oxidases are peroxisomal flavoproteins that catalyze the oxidation of an endo carbon nitrogen bond of N1-acetylspermine in the catabolism of polyamines. While no structure has been reported for a mammalian polyamine oxidase, sequence alignments of polyamine oxidizing flavoproteins identify a conserved histidine residue. Based on the structure of a yeast polyamine oxidase, Saccharomyces cerevisiae Fms1, this residue has been proposed to hydrogen bond to the reactive nitrogen in the polyamine substrate. The corresponding histidine in mouse polyamine oxidase, His64, has been mutated to glutamine, asparagine, and alanine to determine if this residue plays a similar role in the mammalian enzymes. The kinetics of the mutant enzymes were examined with N1-acetylspermine and the slow substrates spermine and N,N'-dibenzyl-1,4-diaminobutane. On average the mutations result in a decrease of ~15-fold in the rate constant for amine oxidation. Rapid-reaction kinetic analyses established that amine oxidation is rate-limiting with spermine as substrate for the wild-type and mutant enzymes and for the H64N enzyme with N1-acetylspermine as substrate. The k(cat)/K(O(2)) value was unaffected by the mutations with N1-acetylspermine as substrate, but decreased ~55-fold with the two slower substrates. The results are consistent with this residue assisting in properly positioning the amine substrate for oxidation.
多胺氧化酶是过氧化物酶体黄素蛋白，可催化多胺分解代谢中 N1-乙酰亚精胺的内碳氮键氧化。虽然尚未报道哺乳动物多胺氧化酶的结构，但多胺氧化黄素蛋白的序列比对鉴定出一个保守的组氨酸残基。基于酿酒酵母 Fms1 的酵母多胺氧化酶结构，该残基被提议与多胺底物中的反应性氮形成氢键。鼠多胺氧化酶中的相应组氨酸 His64 已突变为谷氨酰胺、天冬酰胺和丙氨酸，以确定该残基在哺乳动物酶中是否发挥类似作用。用 N1-乙酰亚精胺和缓慢底物精胺和 N,N'-二苄基-1,4-二氨基丁烷检查突变酶的动力学。突变平均导致胺氧化的速率常数降低约 15 倍。快速反应动力学分析表明，对于野生型和突变酶以及 N1-乙酰亚精胺作为底物的 H64N 酶，胺氧化是限速步骤。对于 N1-乙酰亚精胺作为底物，突变对 k(cat)/K(O(2))值没有影响，但对于两个较慢的底物，k(cat)/K(O(2))值降低了约 55 倍。结果与该残基有助于正确定位氧化的胺底物一致。
