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首页> 外文期刊>Biochemistry >Reaction of Dopa decarboxylase with alpha-methyldopa leads to an oxidative deamination producing 3,4-dihydroxyphenylacetone: An active site directed affinity label
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Reaction of Dopa decarboxylase with alpha-methyldopa leads to an oxidative deamination producing 3,4-dihydroxyphenylacetone: An active site directed affinity label

机译：多巴脱羧酶与α-甲基多巴的反应导致氧化脱氨反应产生3,4-二羟基苯基丙酮：活性位点定向的亲和标记

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摘要

Dopa decarboxylase (DDC) catalyzes the cleavage of alpha-methylDopa into 3,4-dihydroxyphenylacetone and ammonia, via the intermediate alpha-methyldopamine, which does not accumulate during catalysis, The ketone has been identified by high-performance liquid chromatography and mass spectroscopic analysis, and ammonia by means of glutamate dehydrogenase. Molecular oxygen is consumed during the reaction in a 1:2 molar ratio with respect to the products. The k(cat) and K-m of this reaction were determined to be 5.68 min(-1) and 45 mu M, respectively. When the reaction is carried out under anaerobic conditions, alpha-methyldopamine is formed in a time-dependent manner and neither ammonia nor ketone is produced to a significant extent. The reaction is accompanied by a time- and concentration-dependent inactivation of the enzyme with k(inact) of 0.012 min(-1) and K-i of 39.3 mu M. Free 3,4-dihydroxyphenylacetone binds to the active site of DDC and inactivates the enzyme in a time- and concentration-dependent manner with a k(inact)/K-i value similar to that of alpha-methylDopa. D-Dopa, a competitive inhibitor of DDC, protects the enzyme against inactivation. Taken together, these findings indicate the active site directed nature of the interaction of DDC with 3,4-dihydroxyphenylacetone and provide evidence that the ketone generated by the reaction of DDC with alpha-methylDopa dissociates from the active site before it inactivates the enzyme. Inactivation of the enzyme by ketone followed by (NaBH4)-H-3 reduction and chymotryptic digestion revealed that the lysine residue which binds pyridoxal 5'-phosphate (PLP) in the native enzyme is the site of covalent modification. Together with the characterization of the adduct released from the inactivated DDC, these data suggest that the enzyme is inactivated by trapping the coenzyme in a ternary adduct with ketone and the active site lysine. As recently reported for serotonin (5-MT) [Bertoldi, M., Moore, P. S., Maras, B., Dominici, P., and Borri Voltattorni, C. (1996) J. Biol. Chem, 271, 23954-23959], the conversion of dopamine (DA) into 3,4-dihydroxyphenylacetaldehyde and ammonia catalyzed by DDC is accompanied by irreversible loss of decarboxylase activity. However, the comparison between the absorbance, fluorescence, and CD features of DDC after 5-HT- or 3,4-dihydroxyphenylacetone-induced inactivation shows that a different covalent adduct is formed between either of these two molecules and DDC-bound PLP. [References: 28]
机译：多巴脱羧酶（DDC）通过中间体α-甲基多巴胺催化α-甲基多巴裂解为3,4-二羟基苯基丙酮和氨，该酮在催化过程中不会积累。酮已通过高效液相色谱和质谱分析鉴定和氨通过谷氨酸脱氢酶。相对于产物，分子氧在反应期间以1：2的摩尔比被消耗。测定该反应的k（cat）和K-m分别为5.68min（-1）和45μM。当反应在厌氧条件下进行时，α-甲基多巴胺以时间依赖性方式形成，并且氨和酮都不会大量产生。反应伴随时间和浓度的失活，k（inact）为0.012 min（-1），Ki为39.3μM。游离的3,4-二羟基苯丙酮与DDC的活性位结合并失活以与时间和浓度有关的方式产生酶，其ak（inact）/ Ki值类似于α-methylDopa。 DDC的竞争性抑制剂D-Dopa保护该酶免于失活。综上所述，这些发现表明了DDC与3，4-二羟基苯基丙酮的相互作用的活性位点定向性质，并提供了证据，表明由DDC与α-甲基多巴反应生成的酮在使酶失活之前已从活性位点解离。酮使酶失活，然后进行（NaBH4）-H-3还原和胰凝乳蛋白酶消化，发现与天然酶中的吡ido醛5'-磷酸酯（PLP）结合的赖氨酸残基是共价修饰的位点。这些数据与从灭活的DDC释放的加合物的特征一起，表明该酶是通过将辅酶与酮和活性位点赖氨酸一起捕获在三元加合物中而失活的。如最近关于5-羟色胺（5-MT）的报道[Bertoldi，M.，Moore，P.S。，Maras，B.，Dominici，P。，和Borri Voltattorni，C。（1996）J.Biol.Acad.Sci。 Chem，271，23954-23959]，DDC催化多巴胺（DA）转化为3,4-二羟基苯基乙醛和氨会伴随着不可逆的脱羧酶活性损失。但是，在5-HT-或3,4-二羟基苯基丙酮诱导的失活后，DDC的吸光度，荧光和CD特征之间的比较表明，这两个分子之一与DDC结合的PLP之间形成了不同的共价加合物。 [参考：28]

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