Brennan T V, Anderson J W, Jia Z, Waygood E B, Clarke S
Department of Chemistry and Biochemistry, University of California, Los Angeles 90024-1569.
J Biol Chem. 1994 Oct 7;269(40):24586-95.
The non-enzymatic deamidation at residues Asn-12 and Asn-38 of Escherichia coli phosphocarrier protein, HPr, and the repair of the resulting L-isoaspartyl (or beta-aspartyl) derivatives, HPr-1 and HPr-2, by recombinant human S-adenosylmethionine-dependent L-isoaspartate-(D-aspartate) O-methyltransferase (EC 2.1.1.77) were investigated. HPr is a component of the bacterial phosphoenolpyruvate:sugar phosphotransferase system that is involved in the concomitant translocation and phosphorylation of many hexose sugars. The major products of the deamidation reaction, L-isoaspartyl (or beta-aspartyl) residues at positions 12 and 38, were found to be substrates for the L-isoaspartate-(D-aspartate) O-methyltransferase, an enzyme active on a wide variety of peptides and proteins containing these abnormal residues. This enzyme has been shown to catalyze the first step in a process that can convert L-isoaspartyl residues in peptides to normal L-aspartyl residues. The affinity of a recombinant human methyltransferase for HPr-1, a form deamidated at Asn-38, was relatively poor (Km = 3.6 mM), while a greater affinity was found for HPr-2, a form deamidated at both Asn-12 and Asn-38 (Km = 197 microM). When HPr-2 was incubated with S-adenosylmethionine and the methyltransferase, the bulk of the L-isoaspartyl residues at position 12 was converted to L-aspartyl residues. The major-by-product was the D-isoaspartyl form. The conversion of L-isoaspartyl residues at position 38 to L-aspartyl residues was less complete, reflecting the lower affinity of the methyltransferase for this site. The phosphohydrolysis activity of the repaired form was found to be midway between the form containing only L-aspartyl residues at positions 12 and 38 and the deamidated HPr-2 form.
研究了大肠杆菌磷酸载体蛋白HPr的天冬酰胺-12和天冬酰胺-38残基处的非酶促脱酰胺作用，以及重组人S-腺苷甲硫氨酸依赖性L-异天冬氨酸-（D-天冬氨酸）O-甲基转移酶（EC 2.1.1.77）对由此产生的L-异天冬氨酰（或β-天冬氨酰）衍生物HPr-1和HPr-2的修复作用。HPr是细菌磷酸烯醇丙酮酸：糖磷酸转移酶系统的一个组成部分，该系统参与多种己糖的伴随转运和磷酸化。脱酰胺反应的主要产物，即12位和38位的L-异天冬氨酰（或β-天冬氨酰）残基，被发现是L-异天冬氨酸-（D-天冬氨酸）O-甲基转移酶的底物，该酶对多种含有这些异常残基的肽和蛋白质具有活性。已证明该酶催化肽中L-异天冬氨酰残基转化为正常L-天冬氨酰残基过程的第一步。重组人甲基转移酶对在天冬酰胺-38处脱酰胺的HPr-1的亲和力相对较差（Km = 3.6 mM），而对在天冬酰胺-12和天冬酰胺-38处均脱酰胺的HPr-2的亲和力更高（Km = 197 microM）。当HPr-2与S-腺苷甲硫氨酸和甲基转移酶一起孵育时，12位的大部分L-异天冬氨酰残基转化为L-天冬氨酰残基。主要副产物是D-异天冬氨酰形式。38位的L-异天冬氨酰残基向L-天冬氨酰残基的转化不太完全，这反映了甲基转移酶对该位点的亲和力较低。发现修复形式的磷酸水解活性介于仅在12位和38位含有L-天冬氨酰残基的形式和脱酰胺的HPr-2形式之间。
