Heck S D, Faraci W S, Kelbaugh P R, Saccomano N A, Thadeio P F, Volkmann R A
Pfizer Central Research, Groton, CT 06340, USA.
Proc Natl Acad Sci U S A. 1996 Apr 30;93(9):4036-9. doi: 10.1073/pnas.93.9.4036.
Since ribosomally mediated protein biosynthesis is confined to the L-amino acid pool, the presence of D-amino acids in peptides was considered for many years to be restricted to proteins of prokaryotic origin. Unicellular microorganisms have been responsible for the generation of a host of D-amino acid-containing peptide antibiotics (gramicidin, actinomycin, bacitracin, polymyxins). Recently, a series of mu and delta opioid receptor agonists [dermorphins and deltorphins] and neuroactive tetrapeptides containing a D-amino acid residue have been isolated from amphibian (frog) skin and mollusks. Amino acid sequences obtained from the cDNA libraries coincide with the observed dermorphin and deltorphin sequences, suggesting a stereospecific posttranslational amino acid isomerization of unknown mechanism. A cofactor-independent serine isomerase found in the venom of the Agelenopsis aperta spider provides the first major clue to explain how multicellular organisms are capable of incorporating single D-amino acid residues into these and other eukaryotic peptides. The enzyme is capable of isomerizing serine, cysteine, O-methylserine, and alanine residues in the middle of peptide chains, thereby providing a biochemical capability that, until now, had not been observed. Both D- and L-amino acid residues are susceptible to isomerization. The substrates share a common Leu-Xaa-Phe-Ala recognition site. Early in the reaction sequence, solvent-derived deuterium resides solely with the epimerized product (not substrate) in isomerizations carried out in 2H2O. Significant deuterium isotope effects are obtained in these reactions in addition to isomerizations of isotopically labeled substrates (2H at the epimerizeable serine alpha-carbon atom). The combined kinetic and structural data suggests a two-base mechanism in which abstraction of a proton from one face is concomitant with delivery from the opposite face by the conjugate acid of the second enzymic base.
由于核糖体介导的蛋白质生物合成局限于L-氨基酸库，因此多年来人们一直认为肽中D-氨基酸的存在仅限于原核生物来源的蛋白质。单细胞微生物产生了许多含D-氨基酸的肽抗生素（短杆菌肽、放线菌素、杆菌肽、多粘菌素）。最近，一系列μ和δ阿片受体激动剂[皮啡肽和强啡肽]以及含有D-氨基酸残基的神经活性四肽已从两栖动物（青蛙）皮肤和软体动物中分离出来。从cDNA文库获得的氨基酸序列与观察到的皮啡肽和强啡肽序列一致，这表明存在一种机制不明的立体特异性翻译后氨基酸异构化。在黑腹栉足蛛毒液中发现一种不依赖辅因子的丝氨酸异构酶，这为解释多细胞生物如何能够将单个D-氨基酸残基掺入这些及其他真核肽中提供了首个重要线索。该酶能够使肽链中间的丝氨酸、半胱氨酸、O-甲基丝氨酸和丙氨酸残基异构化，从而提供了一种迄今为止尚未观察到的生化能力。D-和L-氨基酸残基都易于异构化。底物共享一个共同的Leu-Xaa-Phe-Ala识别位点。在反应序列早期，在2H₂O中进行的异构化反应中，溶剂衍生的氘仅存在于差向异构化产物（而非底物）中。除了同位素标记底物（在可差向异构化的丝氨酸α碳原子处为2H）的异构化反应外，这些反应还获得了显著的氘同位素效应。动力学和结构数据相结合表明存在一种双碱机制，其中从一个面夺取质子与第二个酶碱的共轭酸从相反面传递质子同时发生。
