Merkler D J, Kulathila R, Ash D E
Analytical Protein and Organic Chemistry Group, Unigene Laboratories, Inc., Fairfield, New Jersey 07004.
Arch Biochem Biophys. 1995 Feb 20;317(1):93-102. doi: 10.1006/abbi.1995.1140.
Peptidylglycine alpha-amidating enzyme catalyzes the two-step conversion of C-terminal glycine-extended peptides to C-terminal alpha-amidated peptides and glyoxylate in a reaction that requires O2, ascorbate and 2 mol of copper per mole of enzyme [Kulathila et al. (1994) Arch. Biochem. Biophys. 311, 191-195]. Peptides with a C-terminal alpha-hydroxyglycine residue are intermediates in the amidation reaction. Benzylhydrazine inactivates the enzymatic conversion of dansyl-Tyr-Val-Gly to dansyl-Tyr-Val-NH2 in a time- and concentration-dependent manner. In contrast, the enzymatic conversion of dansyl-Tyr-Val-alpha-hydroxyglycine to dansyl-Tyr-Val-NH2 is unaffected by benzylhydrazine. The plot of 1/(inactivation rate) vs 1/[benzylhydrazine] is parabolic, indicating that the inactivation results from the interaction of 2 mol of benzylhydrazine per mole of enzyme. EPR spectra obtained from benzylhydrazine inactivation reactions carried out in the presence of a radical trap, alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone, show the formation of a carbon-centered benzyl radical. The benzyl radical most likely results from redox chemistry between benzylhydrazine and the enzyme-bound Cu(II) ions because EPR studies show that enzyme-bound Cu(II) is reduced to Cu(I) in the presence of benzylhydrazine. The kinetic constants for benzylhydrazine as a reductant in the amidation reaction were determined at benzylhydrazine concentrations too low to cause significant enzyme inactivation. Mimosine exhibits mixed inhibition vs benzylhydrazine; however, previous results have shown that benzylhydrazine is competitive vs ascorbate [Miller et al. (1992) Arch. Biochem. Biophys. 298, 380-388]. This change in kinetic mechanism coupled with the nonlinear inactivation kinetics have lead to a proposal that the two enzyme-bound Cu(II) atoms are nonequivalent with respect to their reduction by benzylhydrazine.
肽基甘氨酸α-酰胺化酶催化C端甘氨酸延伸肽两步转化为C端α-酰胺化肽和乙醛酸，该反应需要氧气、抗坏血酸，且每摩尔酶需要2摩尔铜[库拉蒂拉等人（1994年），《生物化学与生物物理学报》，311卷，191 - 195页]。具有C端α-羟基甘氨酸残基的肽是酰胺化反应的中间体。苄肼以时间和浓度依赖的方式使丹磺酰-Tyr-Val-Gly酶促转化为丹磺酰-Tyr-Val-NH₂失活。相比之下，丹磺酰-Tyr-Val-α-羟基甘氨酸向丹磺酰-Tyr-Val-NH₂的酶促转化不受苄肼影响。1/（失活速率）对1/[苄肼]的作图呈抛物线状，表明失活是由于每摩尔酶与2摩尔苄肼相互作用所致。在自由基捕获剂α-(4-吡啶-1-氧化物)-N-叔丁基硝酮存在下进行苄肼失活反应得到的电子顺磁共振光谱显示形成了以碳为中心的苄基自由基。苄基自由基很可能源于苄肼与酶结合的铜(II)离子之间的氧化还原化学作用，因为电子顺磁共振研究表明在苄肼存在下酶结合的铜(II)被还原为铜(I)。在苄肼浓度过低以至于不会导致显著酶失活的情况下，测定了苄肼作为酰胺化反应中还原剂的动力学常数。含羞草碱对苄肼表现出混合抑制作用；然而，先前的结果表明苄肼与抗坏血酸存在竞争关系[米勒等人（1992年），《生物化学与生物物理学报》，298卷，380 - 388页]。这种动力学机制的变化以及非线性失活动力学导致有人提出，两个酶结合的铜(II)原子在被苄肼还原方面是不等价的。
