Francisco W A, Merkler D J, Blackburn N J, Klinman J P
Department of Chemistry, University of California, Berkeley 94720-1460, USA.
Biochemistry. 1998 Jun 2;37(22):8244-52. doi: 10.1021/bi973004y.
The bifunctional peptidylglycine alpha-amidating enzyme catalyzes the C-terminal amidation of glycine-extended peptides. The first enzyme activity, peptidylglycine alpha-hydroxylating monooxygenase, catalyzes the oxygen-, ascorbate-, and copper-dependent formation of alpha-hydroxyglycine derivatives. These are substrates for the second enzyme activity, peptidylamidoglycolate lyase, which catalyzes their breakdown to the corresponding C-terminal amidated peptide and glyoxylate as final products. Kinetic and isotope effect studies were carried out with N-benzoylglycine as a substrate at pH 6.0 using monofunctional and bifunctional monooxygenase activities. Kinetic data indicate an equilibrium ordered mechanism, with hippuric acid binding first followed by oxygen. A potentially important difference between the two monooxygenase activities is that product release occurs more slowly from the bifunctional enzyme, indicating an influence of the lyase domain on release of alpha-hydroxyglycine product to solution. Intrinsic isotope effects for the C-H bond cleavage were measured for the monofunctional form of the enzyme using a double-label tracer method, yielding 10.6 +/- 0.8 and 1.20 +/- 0.03 for the primary and alpha-secondary deuterium intrinsic isotope effects, respectively. These values are identical to previous measurements for the analogous enzyme system, dopamine beta-monooxygenase [Miller, S. M., and Klinman, J. P. (1985) Biochemistry 24, 2114-2127]. The identity of intrinsic isotope effects for peptidylglycine alpha-hydroxylating monooxygenase and dopamine beta-monooxygenase with substrates of comparable reactivity (N-benzoylglycine and dopamine, respectively) extends similarities between the two enzymes significantly beyond sequence homology and cofactor requirements.
双功能肽基甘氨酸α-酰胺化酶催化甘氨酸延伸肽的C末端酰胺化。第一种酶活性，肽基甘氨酸α-羟化单加氧酶，催化氧、抗坏血酸盐和铜依赖性的α-羟基甘氨酸衍生物的形成。这些是第二种酶活性肽基酰胺基乙醇酸裂解酶的底物，该酶催化它们分解为相应的C末端酰胺化肽和乙醛酸作为最终产物。在pH 6.0条件下，以N-苯甲酰甘氨酸为底物，利用单功能和双功能单加氧酶活性进行了动力学和同位素效应研究。动力学数据表明是一种平衡有序机制，马尿酸先结合，然后是氧。两种单加氧酶活性之间一个潜在的重要差异是，双功能酶的产物释放较慢，这表明裂解酶结构域对α-羟基甘氨酸产物释放到溶液中有影响。使用双标记示踪法测量了该酶单功能形式的C-H键裂解的内在同位素效应，一级和α-二级氘内在同位素效应分别为10.6±0.8和1.20±0.03。这些值与之前对类似酶系统多巴胺β-单加氧酶的测量结果相同[米勒，S.M.，和克林曼，J.P.（1985年）《生物化学》24，2114 - 2127]。肽基甘氨酸α-羟化单加氧酶和多巴胺β-单加氧酶在具有可比反应性的底物（分别为N-苯甲酰甘氨酸和多巴胺）上的内在同位素效应相同，这显著扩展了这两种酶之间的相似性，超出了序列同源性和辅因子要求。
